Analysis Report

0 Description of Workflow

Previous studies suggested that de novo assembly is beneficial for significantly differentially expressed genes (DEG) even when a reference genome is available. ^[1] Moreover, it is now widely recognized that performing transcript quantification and afterwards obtaining the gene expression by adding together the expression from the individual transcripts will result in improved gene-level analysis. ^{[2, 3]} Therefore, the current analysis for this bulk RNA-seq data set is based on the transcript-level with de novo transcriptome assembly. The current analysis protocol was modified from a guideline attached to a peer-reviewed bioinformatic software called IsoformSwitchAnalyzeR ^[4] (Please Click HERE to check the origianl protocol). Briefly, the current protocol can be described as a 6-step process:

1. After QC and potential quality trimming, the reads were mapped to the genome with the STAR aligner.

2. Then the de novo transcriptome assembly was performed with StringTie software and was guided by existing annoation from ENSEMBL database.

3. The third step is to merge the assembled transcripts from all individual StringTie runs into one combined reference transcriptome with StringTie software. The merged GTF file was further annotated with SQANTI3 software, which is designed to annotate the data generated from oxford nanopore sequencing. Meanwhile, the Circular RNA was detected with CIRCexplorer2 software ^[5] from the mapping results created in Step 1 based on the merged GTF file.

4. To make the analysis of the individual samples comparable, all samples were re-quantified using the merged GTF file created in Step 3 with a quasi-mapping aligner called Salmon.

5. Applying a “rescue” algorithm embbed in the IsoformSwitchAnalyzeR ^[4] software to the merged GTF file, to ensures that all novel isoforms are assigned to genes and that “reference gene_id” and “reference gene_names” are used instead of “Stringtie gene_is” for all already annotated genes.The “rescued” GTF file was annotated by SQANTI3 software again and the still remained novel genes (“Stringtie genes” not overlapping any “Refrence genes”) was removed for the downstream analysis.

6. Finally, after correction of batch effect by RUVSeq software, the significant differentially-expressed genes (DEGs) were detected by DESeq2 software; the significant differential exon usage (DEU) were detected by DEXseq software; the analysis of isoform switches with functional consequences and the associated alternative splicing was performed using IsoformSwitchAnalyzeR software; the significant differentially-expressed circular RNAs were detected by circRNAprofiler software.

The source code is avaible on the Supplementary section to reproduce this analysis.

In addition, other data set was generated in order to use the leafcutter software to check the alternative splicing at nucleotide level; to use the Ularcirc software to visulize, to predict ORF, and to estimate the potenial biological function of detected circular RNAs.

[1] Wang S, Gribskov M. Comprehensive evaluation of de novo transcriptome assembly programs and their effects on differential gene expression analysis. Bioinformatics. 2017 Feb 1;33(3):327-33.

[2] Soneson C, Love MI, Robinson MD. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. 2015;4.

[3] Yi L, Pimentel H, Bray NL, Pachter L. Gene-level differential analysis at transcript-level resolution. Genome biology. 2018 Dec;19(1):1-1.

[4] Vitting-Seerup K, Sandelin A. IsoformSwitchAnalyzeR: analysis of changes in genome-wide patterns of alternative splicing and its functional consequences. Bioinformatics. 2019 Nov 1;35(21):4469-71.

[5] Zhang XO, Dong R, Zhang Y, Zhang JL, Luo Z, Zhang J, Chen LL, Yang L. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs. Genome research. 2016 Sep 1;26(9):1277-87.

1 Sample Sequencing Statistics

1.1 Reference

Annotation Files

• Genome sequence reference:
  • Mus_musculus.GRCm39.dna.primary_assembly.fa.gz
    
    
• Coding sequence reference:
  • .gz
    
    
• Non-coding RNA reference:
  • .gz
    
    
• Annotation of features (Origin):
  • Mus_musculus.GRCm39.109.gtf.gz
    
    
• Annotation of features (De Novo Assembly):
  • DeNovoAssemblyTranscriptome.gtf.gz
    
    
• BAM files from STAR aligner which used for
  de novo transcriptome assembly:
  • BAM_Files.zip

Help

The de novo assembly transcriptome and mapping results could be visualized using IGV software. The detials could be checked below:

1.2 Quality Control Report

Click HERE to check the FastQC results.

Click HERE to inspect the de novo transcriptome assembly results.

1.3 Quasi-Mapping by Salmon software

*Mapping Rate (%): the number of reads mapped on genome features (i.e. a genome region contains genes)

Automatically detected most likely library type as U.

Click HERE to learn more detials about the fragment library types.

1.4 Correction of Batch Effect by RUVSeq

Before Correction

RLE plot

RLE plot: Relative log expression plots.

PCA plot

After Correction

RLE plot

RLE plot: Relative log expression plots.

PCA plot

2 Analysis

2.1 Basic Information

Experiment Design

Name	Group	Numbering	Description
MCC1	Control	MCC1	Control
MCC2	Control	MCC2	Control
MCC3	Control	MCC3	Control
MCK1	shVgll3	MCK1	shVgll3
MCK2	shVgll3	MCK2	shVgll3
MCK3	shVgll3	MCK3	shVgll3

Parameter Setting

• The genes with read number > 10 in at least 3 samples were defined as expressed genes.
• Differentially Expressed Genes (DEGs) were defined as FDR < 0.05 and |log2FC| > 1.
• DEGs testing method in DESeq2 software was “LRT”.
• Hierarchical cluster analysis for PCA and Heatmap plots was “ward.D2” based on “pearson” distance.
• Significantly enriched GO terms in GSEA analysis were defined as P-value < 0.05.
• Significantly enriched KEGG terms in GSEA analysis were defined as P-value < 0.05.
• Significantly enriched GO terms in Over Representation Analysis were defined as P-value < 0.05.
• Significantly enriched KEGG terms in Over Representation Analysis analysis were defined as P-value < 0.05.
• Transcripts with isoform fraction (IF) > 5% were defined as expressed transcripts (only validate when using IsoformSwitchAnalyzeR).
• Significant isoform switching were defined as FDR < 0.05 and dIf > 0.05.
• Differentially Expressed Exons were defined as FDR < 0.1.
• Differential intron excision in leafcutter software were defined as FDR < 0.05.

2.2 Differential Gene Expression Analysis

Click HERE to check the read counts, TPM, and feature length of all mapped genes across all samples in a Microsoft .excel file. Click HERE to check all parameters of DESeq2 model for all detected genes of all samples in a Microsoft .excel file. Click HERE to check the DESeq2 model estimated mean gene expression level of each condition (the intercept of a linear model) and its standard errors. For more detials of the DESeq2 model, please Click HERE.

Overview of Differentially Expressed Genes (DEGs)
									Sig. AS
					Sig. DEGs								in sig. DEGs
	All Detected Genes	Non Sig. DEGs			Up- DEGs	Down- DEGs			Genes with Sig. AS	in Non Sig. DEGs			in Up- DEGs	in Down- DEGs
shVgll3 vs Control	15232	14139			520	573			582	515			32	35
† TPM, Transcripts Per Kilobase Million; DEGs, Differentially-Expressed Genes; Sig., Significant; Up-DEGs, Significantly Up-Regulated Genes; Down-DEGs, Significantly Down-Regulated Genes; AS, Alternative Splicing

2.3 Functional Enrichment Analysis

Number of Significantlly-Enriched Terms of Functional Enrichment Analysis (FEA)
	GSEA						ORA
							GO-BP					GO-MF					GO-CC					KEGG
	BP	MF	CC	KEGG			From All Sig. DEGs	From Up- DEGs	From Down- DEGs			From All Sig. DEGs	From Up- DEGs	From Down- DEGs			From All Sig. DEGs	From Up- DEGs	From Down- DEGs			From All Sig. DEGs	From Up- DEGs	From Down- DEGs
shVgll3 vs Control	240	55	54	14			1635	839	1250			119	77	91			100	61	66			48	21	43
† GSEA, Gene Set Enrichment Analysis; ORA, Over Representation Analysis; DEGs, Differentially-Expressed Genes; GO-BP, Gene Ontology Biological Processes; GO-MF, Gene Ontology Molecular Function; GO-CC, Gene Ontology Cellular Component; KEGG, Kyoto Encyclopedia of Genes and Genomes; Sig., Significant; Up-DEGs, Significantly Up-Regulated Genes; Down-DEGs, Significantly Down-Regulated Genes

2.4 Analysis of Isoform Switches

Click HERE to download all results of the significantly-switched isoforms analysis (q < 0.05 and dIF > 0.05).

Click HERE to download the alternative splicing analysis results for visualization by LeafCutter software.

Alternative Splicing Events

Amount of Event

Enrichment Analysis

Comparison Analysis

Abbreviations

• IR : Intron Retention.
• A5 : Alternative 5’ donor site (changes in the 5’end of the upstream exon).
• A3 : Alternative 3’ acceptor site (changes in the 3’end of the downstream exon).
• ATSS : Alternative Transcription Start Site.
• ATTS : Alternative Transcription Termination Site.
• ES : Exon Skipping (EI means Exon Inclusion).
• MES : Multiple Exon Skipping. Skipping of >1 consecutive exons. (MEI means Multiple Exon Inclusion).
• MEE : Mutually Exclusive Exons.

Biological Consequences

Amount of Event

Enrichment Analysis

Comparison Analysis

Abbreviations

• tss : Test transcripts for whether they use different Transcription Start Site (TSS) (more than ntCutoff from each other).
• tts : Test transcripts for whether they use different Transcription Termination Site (TTS) (more than ntCutoff from each other).
• last_exon : Test whether transcripts utilizes different last exons (defined as the last exon of each transcript is non-overlapping).
• isoform_seq_similarity : Test whether the isoform nucleotide sequences are different (as described above). Reported as different if the measured JCsim is smaller than ntJCsimCutoff and the length difference of the aligned and combined region is larger than ntCutoff.
• isoform_length : Test transcripts for differences in isoform length. Only reported if the difference is larger than indicated by the ntCutoff and ntFracCutoff. Please * note that this is a less powerful analysis than implemented in ‘isoform_seq_similarity’ as two equally long sequences might be very different.
• exon_number : Test transcripts for differences in exon number.
• intron_structure : Test transcripts for differences in intron structure, e.g. usage of exon-exon junctions. This analysis corresponds to analyzing whether all introns in one isoform is also found in the other isoforms.
• intron_retention : Test for differences in intron retentions (and their genomic positions). Require that analyzeIntronRetention have been run.
• isoform_class_code : Test transcripts for differences in the transcript classification provide by cufflinks. For a updated list of class codes see http://cole-trapnell-lab.github.io/cufflinks/cuffcompare/#transfrag-class-codes.
• coding_potential : Test transcripts for differences in coding potential, as indicated by the CPAT or CPC2 analysis. Requires that analyzeCPAT or analyzeCPC2 have been used to add external conding potential analysis to the switchAnalyzeRlist.
• ORF_seq_similarity : Test whether the amino acid sequences of the ORFs are different (as described above). Reported as different if the measured JCsim is smaller than AaJCsimCutoff and the length difference of the aligned and combined region is larger than AaCutoff. Requires that least one of the isoforms are annotated with a ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• ORF_genomic : Test transcripts for differences in genomic position of the Open Reading Frames (ORF). Requires that least one of the isoforms are annotated with an ORF either via identifyORF or by supplying a GTF file and settingaddAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• ORF_length : Test transcripts for differences in length of Open Reading Frames (ORF). Note that this is a less powerful analysis than implemented in ORF_seq_similarity as two equally long sequences might be very different. Only reported if the difference is larger than indicated by the ntCutoff and ntFracCutoff. Requires that least one of the isoforms are annotated with a ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• 5_utr_seq_similarity : Test whether the isoform nucleotide sequences of the 5’ UnTranslated Region (UTR) are different (as described above). The 5’UTR is defined as the region from the transcript start to the ORF start. Reported as different if the measured JCsim is smaller than ntJCsimCutoff and the length difference of the aligned and combined region is larger than ntCutoff. Requires that both the isoforms are annotated with an ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• 5_utr_length : Test transcripts for differences in the length of the 5’ UnTranslated Region (UTR). The 5’UTR is defined as the region from the transcript start to the ORF start. Note that this is a less powerful analysis than implemented in ‘5_utr_seq_similarity’ as two equally long sequences might be very different. Only reported if the difference is larger than indicated by the ntCutoff and ntFracCutoff. Requires that both the isoforms are annotated with a ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• 3_utr_seq_similarity : Test whether the isoform nucleotide sequences of the 3’ UnTranslated Region (UTR) are different (as described above). The 3’UTR is defined as the region from the end of the ORF to the transcript end. Reported as different if the measured JCsim is smaller than ntJCsimCutoff and the length difference of the aligned and combined region is larger than ntCutoff. Requires that both the isoforms are annotated with a ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• 3_utr_length : Test transcripts for differences in the length of the 3’ UnTranslated Regions (UTR). The 3’UTR is defined as the region from the end of the ORF to the transcript end. Note that this is a less powerful analysis than implemented in 3_utr_seq_similarity as two equally long sequences might be very different. Requires that identifyORF have been used to predict NMD sensitivity or that the ORF was imported though one of the dedicated import functions implemented in isoformSwitchAnalyzeR. Only reported if the difference is larger than indicated by the ntCutoff and ntFracCutoff. Requires that both the isoforms are annotated with a ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• NMD_status : Test transcripts for differences in sensitivity to Nonsense Mediated Decay (NMD). Requires that both the isoforms have been annotated with PTC either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• domains_identified : Test transcripts for differences in the name and order of which domains are identified by the Pfam in the transcripts. Requires that analyzePFAM have been used to add external Pfam analysis to the switchAnalyzeRlist. Requires that both the isoforms are annotated with a ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• domain_length : Test transcripts for differences in the length of overlapping domains of the same type (same hmm_name) thereby enabling analysis of protein domain truncation. Do however note that a small difference in length is will likely not truncate the protein domain. The length difference, measured in AA, must be larger than AaCutoff and AaFracCutoff .Requires that analyzePFAM have been used to add external Pfam analysis to the switchAnalyzeRlist. Requires that both the isoforms are annotated with a ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist.
• genomic_domain_position : Test transcripts for differences in the genomic position of the domains identified by the Pfam analysis (Will be different unless the two isoforms have the same domains at the same genomic location). Requires that analyzePFAM have been used to add external Pfam analysis to the switchAnalyzeRlist. Requires that both the isoforms are annotated with a ORF either via identifyORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist (and are thereby also affected by removeNoncodinORFs=TRUE in analyzeCPAT).
• IDR_identified : Test for differences in isoform IDRs. Specifically the two isoforms are analyzed for whether they contain IDRs which do not overlap in genomic coordinates. Requires that analyzeNetSurfP2 or analyzeIUPred2A have been used to add external IDR analysis to the switchAnalyzeRlist.
• IDR_length : Test for differences in the length of overlapping (in genomic coordinates) IDRs. The length difference, measured in AA, must be larger than AaCutoff and AaFracCutoff. Requires that analyzeNetSurfP2 or analyzeIUPred2A have been used to add external IDR analysis to the switchAnalyzeRlist.
• IDR_type : Test for differences in IDR type. Specifically the two isoforms are tested for overlapping IDRs (genomic coordinates) and overlapping IDRs are compared with regards to their IDR type (IDR vs IDR w binding site). Only available if analyzeIUPred2A was used to add external IDR analysis to the switchAnalyzeRlist.
• signal_peptide_identified : Test transcripts for differences in whether a signal peptide was identified or not by the SignalP analysis. Requires that analyzeSignalP have been used to add external SignalP analysis to the switchAnalyzeRlist. Requires that both the isoforms are annotated with a ORF either via analyzeORF or by supplying a GTF file and setting addAnnotatedORFs=TRUE when creating the switchAnalyzeRlist (and are thereby also affected by removeNoncodinORFs=TRUE in analyzeCPAT).

Volcano Plot

Help

Please Click HERE to learn more details about the results of IsoformSwitchAnalyzeR.

2.4 Circular RNA Analysis

Spreadsheet

Click HERE to download the circRNA analysis results for visualization by Ularcirc software. For using the Ularcirc software, please also download and install the corresponding BSgenome, TxDb, and annotation packages in your R enviroment.

id	gene	allTranscripts	transcript	totExons	strand	chrom	startUpIntron	endUpIntron	startUpBSE	endUpBSE	startDownBSE	endDownBSE	startDownIntron	endDownIntron	exNumUpBSE	exNumDownBSE	numOfExons	lenUpIntron	lenUpBSE	lenDownBSE	lenDownIntron	meanLengthBSEs	meanLengthIntrons	lenCircRNA	Read # on BSJ
ENSMUSG00000029661:+:6:4529067:4531010	ENSMUSG00000029661	ENSMUST00000031668	ENSMUST00000031668	52	+	6	4528414	4529067	4529068	4529121	4530957	4531010	4531011	4531103	33	35	3	653	53	53	92	53	372.5	162	6
ENSMUSG00000001506:+:11:94840922:94841030	ENSMUSG00000001506	ENSMUST00000001547,MSTRG.4128.2,MSTRG.4128.6,MSTRG.4128.3	ENSMUST00000001547	51	+	11	94840600	94840922	94840923	94841030	94840923	94841030	94841031	94841116	47	47	1	322	107	107	85	107	203.5	108	3
ENSMUSG00000029661:+:6:4521395:4522495	ENSMUSG00000029661	ENSMUST00000031668,ENSMUST00000148864	ENSMUST00000031668	52	+	6	4521285	4521395	4521396	4521494	4522442	4522495	4522496	4522955	23	24	2	110	98	53	459	75.5	284.5	153	3
ENSMUSG00000048126:-:1:90720735:90720798	ENSMUSG00000048126	MSTRG.815.3,ENSMUST00000056925,MSTRG.815.4,ENSMUST00000097653,ENSMUST00000188587,ENSMUST00000136916	MSTRG.815.3	44	-	1	90721731	90720799	90720798	90720736	90720798	90720736	90720735	90720198	21	21	1	932	62	62	537	62	734.5	63	2
ENSMUSG00000029661:+:6:4535380:4535884	ENSMUSG00000029661	ENSMUST00000031668	ENSMUST00000031668	52	+	6	4534689	4535380	4535381	4535488	4535831	4535884	4535885	4535975	42	43	2	691	107	53	90	80	390.5	162	1
ENSMUSG00000057897:-:11:5929672:5932572	ENSMUSG00000057897	ENSMUST00000109813,ENSMUST00000090443,ENSMUST00000109815,ENSMUST00000155755,ENSMUST00000019133,ENSMUST00000093355,ENSMUST00000109812,ENSMUST00000002817,MSTRG.3100.12,ENSMUST00000101586	ENSMUST00000109813	21	-	11	5932720	5932573	5932572	5932501	5929721	5929673	5929672	5922889	15	17	3	147	71	48	6783	59.5	3465	166	1
ENSMUSG00000089945:+:4:57854561:57857037	ENSMUSG00000089945	MSTRG.15990.3,MSTRG.15990.4,MSTRG.15990.2,MSTRG.15990.5,ENSMUST00000098066,ENSMUST00000150412	MSTRG.15990.3	10	+	4	57757684	57854561	57854562	57857037	57854562	57857037	57857038	57883023	8	8	1	96877	2475	2475	25985	2475	61431	2476	1
ENSMUSG00000028926:-:5:5060832:5153854	ENSMUSG00000028926	ENSMUST00000115451,ENSMUST00000030763,ENSMUST00000133465,ENSMUST00000115450,ENSMUST00000115452	ENSMUST00000115451	14	-	5	5185318	5153855	5153854	5153792	5060926	5060833	5060832	4999596	6	9	4	31463	62	93	61236	77.5	46349.5	402	1
ENSMUSG00000022443:-:15:77647421:77647530	ENSMUSG00000022443	ENSMUST00000016771,MSTRG.8234.2	ENSMUST00000016771	41	-	15	77647991	77647531	77647530	77647422	77647530	77647422	77647421	77647325	39	39	1	460	108	108	96	108	278	109	1
ENSMUSG00000032925:+:14:124077974:124094991	ENSMUSG00000032925	ENSMUST00000049681,ENSMUST00000142161,MSTRG.7733.4,MSTRG.7733.1,ENSMUST00000132026	ENSMUST00000049681	11	+	14	124065311	124077974	124077975	124078097	124094845	124094991	124094992	124191982	4	7	4	12663	122	146	96990	134	54826.5	552	1
ENSMUSG00000029661:+:6:4532743:4532797	ENSMUSG00000029661	ENSMUST00000031668,ENSMUST00000155687	ENSMUST00000031668	52	+	6	4531949	4532743	4532744	4532797	4532744	4532797	4532798	4533741	39	39	1	794	53	53	943	53	868.5	54	1
ENSMUSG00000040998:-:3:132610418:132623314	ENSMUSG00000040998	ENSMUST00000042744,ENSMUST00000117811,ENSMUST00000042729,ENSMUST00000117456,ENSMUST00000093971,ENSMUST00000117164	ENSMUST00000042744	12	-	3	132653807	132623315	132623314	132623222	132610814	132610419	132610418	132597250	3	8	6	30492	92	395	13168	243.5	21830	987	1
ENSMUSG00000029761:+:6:34639062:34639151	ENSMUSG00000029761	MSTRG.18965.6,MSTRG.18965.4,ENSMUST00000115027,MSTRG.18965.1,ENSMUST00000079391,ENSMUST00000115026,ENSMUST00000155714,ENSMUST00000154182	MSTRG.18965.6	14	+	6	34575601	34639062	34639063	34639151	34639063	34639151	34639152	34662897	2	2	1	63461	88	88	23745	88	43603	89	1
ENSMUSG00000052698:-:9:67367197:67367271	ENSMUSG00000052698	ENSMUST00000040025,ENSMUST00000215784,ENSMUST00000039662	ENSMUST00000040025	58	-	9	67466895	67367272	67367271	67367198	67367271	67367198	67367197	67304966	2	2	1	99623	73	73	62231	73	80927	74	1
ENSMUSG00000060992:+:15:103205160:103205212	ENSMUSG00000060992	ENSMUST00000230893,ENSMUST00000100162	ENSMUST00000230893	8	+	15	103205038	103205160	103205161	103205212	103205161	103205212	103205213	103206313	7	7	1	122	51	51	1100	51	611	52	1
ENSMUSG00000014850:-:13:92435660:92436866	ENSMUSG00000014850	ENSMUST00000191550,ENSMUST00000185852,ENSMUST00000022220	ENSMUST00000191550	25	-	13	92445952	92436867	92436866	92436782	92435770	92435661	92435660	92422518	12	13	2	9085	84	109	13142	96.5	11113.5	195	1
ENSMUSG00000038954:+:17:45097496:45097597	ENSMUSG00000038954	ENSMUST00000130623,ENSMUST00000050630,ENSMUST00000127798	ENSMUST00000130623	3	+	17	45088316	45097496	45097497	45097597	45097497	45097597	45097598	45234065	2	2	1	9180	100	100	136467	100	72823.5	101	1
ENSMUSG00000001424:+:6:28626098:28795937	ENSMUSG00000001424	ENSMUST00000167201,ENSMUST00000164915,ENSMUST00000001460	ENSMUST00000167201	17	+	6	28545598	28626098	28626099	28626188	28795828	28795937	28795938	28933365	11	16	6	80500	89	109	137427	99	108963.5	627	1
ENSMUSG00000026918:-:2:27342844:27344516	ENSMUSG00000026918	ENSMUST00000028282,ENSMUST00000113941,ENSMUST00000077737,MSTRG.12589.3,ENSMUST00000164296,ENSMUST00000154316,ENSMUST00000138693	ENSMUST00000028282	12	-	2	27346920	27344517	27344516	27344325	27343086	27342845	27342844	27340562	8	9	2	2403	191	241	2282	216	2342.5	434	1
ENSMUSG00000040990:+:X:158547007:158586819	ENSMUSG00000040990	ENSMUST00000112456,ENSMUST00000073094,ENSMUST00000141354,ENSMUST00000080394,ENSMUST00000150433,ENSMUST00000123433	ENSMUST00000112456	18	+	X	158466698	158547007	158547008	158547131	158586716	158586819	158586820	158599572	3	4	2	80309	123	103	12752	113	46530.5	228	1
ENSMUSG00000029661:+:6:4531237:4532797	ENSMUSG00000029661	ENSMUST00000031668,ENSMUST00000155687	ENSMUST00000031668	52	+	6	4531158	4531237	4531238	4531345	4532744	4532797	4532798	4533741	37	39	3	79	107	53	943	80	511	216	1
ENSMUSG00000004980:-:6:51441068:51441191	ENSMUSG00000004980	MSTRG.19150.9,MSTRG.19150.8,MSTRG.19150.7,MSTRG.19150.11,MSTRG.19150.5,ENSMUST00000114459,ENSMUST00000203220,ENSMUST00000203954,ENSMUST00000090002,ENSMUST00000069949,ENSMUST00000204188,ENSMUST00000204158,MSTRG.19150.10,MSTRG.19150.13,MSTRG.19150.12,ENSMUST00000204090,ENSMUST00000203253	MSTRG.19150.9	10	-	6	51441407	51441192	51441191	51441069	51441191	51441069	51441068	51440474	9	9	1	215	122	122	594	122	404.5	123	1
ENSMUSG00000001119:-:10:76554206:76555510	ENSMUSG00000001119	ENSMUST00000001147	ENSMUST00000001147	35	-	10	76556794	76555511	76555510	76555457	76554251	76554207	76554206	76553947	9	13	5	1283	53	44	259	48.5	771	198	1
ENSMUSG00000034341:-:11:115970533:115971170	ENSMUSG00000034341	ENSMUST00000074628,ENSMUST00000106444,ENSMUST00000145278	ENSMUST00000074628	8	-	11	115971371	115971171	115971170	115971048	115970610	115970534	115970533	115970423	6	7	2	200	122	76	110	99	155	200	1
ENSMUSG00000008333:+:2:142911293:142911344	ENSMUSG00000008333	ENSMUST00000008477,ENSMUST00000134091,ENSMUST00000126763,ENSMUST00000127760	ENSMUST00000008477	7	+	2	142910322	142911293	142911294	142911344	142911294	142911344	142911345	142912807	5	5	1	971	50	50	1462	50	1216.5	51	1
ENSMUSG00000029763:+:6:33324717:33557013	ENSMUSG00000029763	ENSMUST00000052266,ENSMUST00000132842	ENSMUST00000052266	18	+	6	33309390	33324717	33324718	33324892	33556917	33557013	33557014	33734901	7	10	4	15327	174	96	177887	135	96607	507	1
ENSMUSG00000034771:+:10:81416090:81416165	ENSMUSG00000034771	ENSMUST00000146358,ENSMUST00000146916,ENSMUST00000136341,ENSMUST00000143285	ENSMUST00000146358	21	+	10	81413526	81416090	81416091	81416165	81416091	81416165	81416166	81416317	6	6	1	2564	74	74	151	74	1357.5	75	1
ENSMUSG00000091509_ENSMUSG00000022119:-:14:105367668:105367836	ENSMUSG00000091509_ENSMUSG00000022119	MSTRG.7680.4,MSTRG.7680.3,MSTRG.7680.5,MSTRG.7680.1	MSTRG.7680.4	23	-	14	105368918	105367837	105367836	105367669	105367836	105367669	105367668	105366106	17	17	1	1081	167	167	1562	167	1321.5	168	1

Help

Please Click HERE to learn more details about the results of circRNAprofiler.

Please Click HERE to learn more details for using the Ularcirc software.

The below video shows the using of Ularcirc software.

3 Support

We work hard to ensure that this wrapped script is a powerful tool empowering your research. However, no software is free of bugs and issues, therefore we would love to get feedback from our users.

The below video is a brief introduction of the basic elements of this analysis report.

4 Citation

If you find this code useful in your research, please cite:

Not yet available

5 Supplementary

S1. Source Code

Please check my personal GitHub to find the source code.

S2. Running log

Welcome to Ubuntu 22.04.2 LTS (GNU/Linux 5.19.0-35-generic x86_64)

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14 updates can be applied immediately. To see these additional updates run: apt list –upgradable

20 additional security updates can be applied with ESM Apps. Learn more about enabling ESM Apps service at https://ubuntu.com/esm

20 updates could not be installed automatically. For more details, see /var/log/unattended-upgrades/unattended-upgrades.log

[2023年 3月 7日 火曜日 14:29:19 JST] Create folder for FastQC of origin and filterd fastq files

[2023年 3月 7日 火曜日 14:29:19 JST] Create folder for FastQC of bam files from STAR

[2023年 3月 7日 火曜日 14:29:19 JST] Download genome reference and unzip it

[2023年 3月 7日 火曜日 14:32:26 JST] Download genome annotation and unzip it Writing new genes GTF file (may take 10 minutes for a 1GB input GTF file)… …done

Reading in genome_filtered.gtf Processing… Warning: there are empty ‘gene_name’ attributes, using ‘gene_id’ for them Saving exons to genome_filtered_exons.txt

[2023年 3月 7日 火曜日 14:34:36 JST] Downloading Pfam-A.hmm Working… done. Pressed and indexed 19632 HMMs (19632 names and 19632 accessions). Models pressed into binary file: ./Pfam_database/Pfam-A.hmm.h3m SSI index for binary model file: ./Pfam_database/Pfam-A.hmm.h3i Profiles (MSV part) pressed into: ./Pfam_database/Pfam-A.hmm.h3f Profiles (remainder) pressed into: ./Pfam_database/Pfam-A.hmm.h3p

[2023年 3月 7日 火曜日 14:36:52 JST] Did not find sample list file, now create it and check the extension of FASTQ files. The extension of all FASTQ files in this folder will be changed to .fastq

[2023年 3月 7日 火曜日 14:36:52 JST] Extension chekced for ./MCC1_1.fastq.gz, process to the next.

[2023年 3月 7日 火曜日 14:36:52 JST] Extension chekced for ./MCC2_1.fastq.gz, process to the next.

[2023年 3月 7日 火曜日 14:36:52 JST] Extension chekced for ./MCC3_1.fastq.gz, process to the next.

[2023年 3月 7日 火曜日 14:36:52 JST] Extension chekced for ./MCK1_1.fastq.gz, process to the next.

[2023年 3月 7日 火曜日 14:36:52 JST] Extension chekced for ./MCK2_1.fastq.gz, process to the next.

[2023年 3月 7日 火曜日 14:36:52 JST] Extension chekced for ./MCK3_1.fastq.gz, process to the next.

[2023年 3月 7日 火曜日 14:36:52 JST] FastQC of origin fastq files

Analysis complete for MCC1_1.fastq.gz Analysis complete for MCC2_1.fastq.gz Analysis complete for MCK2_1.fastq.gz Analysis complete for MCC3_1.fastq.gz Analysis complete for MCK1_1.fastq.gz Analysis complete for MCK3_1.fastq.gz

[2023年 3月 7日 火曜日 14:37:51 JST] Check FastQC results ./MCC1_1.fastq.gz [2023年 3月 7日 火曜日 14:37:51 JST] READ_LENGTH: 50 [2023年 3月 7日 火曜日 14:37:51 JST] ADAPTER_QC: pass [2023年 3月 7日 火曜日 14:37:51 JST] READ_QC: pass

[2023年 3月 7日 火曜日 14:37:51 JST] DO_QC: 1

[2023年 3月 7日 火曜日 14:37:51 JST] Some of your fastq files have adapter contamination or reads with too low quality. [2023年 3月 7日 火曜日 14:37:51 JST] Create folder for FastQC of qulity trimmed fastq files

[2023年 3月 7日 火曜日 14:37:51 JST] Did not find STAR index, now building STAR index: STAR –runThreadN 128 –runMode genomeGenerate –genomeDir ./GenomeIndex_ENSEMBL_STAR_ReadLength49/ –genomeFastaFiles genome.fa –sjdbGTFfile genome_filtered.gtf –sjdbOverhang 49 –limitGenomeGenerateRAM 208188325888 STAR version: 2.7.10b compiled: 2022-11-01T09:53:26-04:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 07 14:37:51 ….. started STAR run Mar 07 14:37:51 … starting to generate Genome files Mar 07 14:38:19 ….. processing annotations GTF Mar 07 14:38:35 … starting to sort Suffix Array. This may take a long time… Mar 07 14:38:46 … sorting Suffix Array chunks and saving them to disk… Mar 07 14:42:48 … loading chunks from disk, packing SA… Mar 07 14:43:43 … finished generating suffix array Mar 07 14:43:43 … generating Suffix Array index Mar 07 14:46:41 … completed Suffix Array index Mar 07 14:46:41 ….. inserting junctions into the genome indices Mar 07 14:47:50 … writing Genome to disk … Mar 07 14:47:51 … writing Suffix Array to disk … Mar 07 14:48:02 … writing SAindex to disk Mar 07 14:48:04 ….. finished successfully

[2023年 3月 7日 火曜日 14:48:04 JST] Building salmon index **************** *** getDecoy ************* -j = 128 -a = /mnt/disk2/Yuan_Vgl3KO_ResOut/genome_filtered.gtf -g = /mnt/disk2/Yuan_Vgl3KO_ResOut/genome.fa -t = /mnt/disk2/Yuan_Vgl3KO_ResOut/genomeTx.fa -o

= decoys [1/10] Extracting exonic features from the gtf [2/10] Masking the genome fasta [3/10] Aligning transcriptome to genome >>>>>>>>>>>>>>>>>> Reference = [reference.masked.genome.fa] Query = [/mnt/disk2/Yuan_Vgl3KO_ResOut/genomeTx.fa] Kmer size = 16 Window size = 5 Segment length = 500 (read split allowed) Alphabet = DNA Percentage identity threshold = 80% Mapping output file = mashmap.out Filter mode = 1 (1 = map, 2 = one-to-one, 3 = none) Execution threads = 128 >>>>>>>>>>>>>>>>>> INFO, skch::Sketch::build, minimizers picked from reference = 843072471 INFO, skch::Sketch::index, unique minimizers = 276490023 INFO, skch::Sketch::computeFreqHist, Frequency histogram of minimizers = (1, 141625359) … (2606602, 1) INFO, skch::Sketch::computeFreqHist, With threshold 0.001%, ignore minimizers occurring >= 7371 times during lookup. INFO, skch::main, Time spent computing the reference index: 223.128 sec INFO, skch::Map::mapQuery, [count of mapped reads, reads qualified for mapping, total input reads] = [118333, 118505, 149435] INFO, skch::main, Time spent mapping the query : 420.168 sec INFO, skch::main, mapping results saved in : mashmap.out [4/10] Extracting intervals from mashmap alignments [5/10] Merging the intervals [6/10] Extracting sequences from the genome [7/10] Concatenating to get decoy sequences [8/10] Making gentrome [9/10] Extracting decoy sequence ids [10/10] Removing temporary files Threads = 128 Vertex length = 31 Hash functions = 5 Filter size = 4294967296 Capacity = 2 Files: salmon_index/ref_k31_fixed.fa ——————————————————————————– Round 0, 0:4294967296 Pass Filling Filtering 1 14 11
2 11 0 True junctions count = 1203289 False junctions count = 2589034 Hash table size = 3792323 Candidate marks count = 15670767 ——————————————————————————– Reallocating bifurcations time: 0 True marks count: 11958622 Edges construction time: 13 ——————————————————————————– Distinct junctions = 1203289

for info, total work write each : 2.331 total work inram from level 3 : 4.322 total work raw : 25.000 Bitarray 1287077120 bits (100.00 %) (array + ranks ) final hash 0 bits (0.00 %) (nb in final hash 0)

[2023年 3月 7日 火曜日 15:10:14 JST] This folder has 6 fastq files. |—-Now start mapping and counting using STAR and featureCounts [2023年 3月 7日 火曜日 15:10:14 JST] Please Check Read file: ./MCC1_1.fastq.gz

[2023年 3月 7日 火曜日 15:10:14 JST] Please Check Output Folder:./MCC1 The files ./MCC1_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 15:10:14 JST] Run trim_galore pigz 2.6

[2023年 3月 7日 火曜日 15:10:45 JST] FastQC of trimmed fastq files ./fastqc_results_trimmed/./MCC1_1_trimmed_fastqc.html Analysis complete for MCC1_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:11:40 JST] Using Salmon to Map sample 1 with 128 CPU cores: ./MCC1_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:12:35 JST] Mapping sample 1 with 128 CPU cores: ./MCC1_1_trimmed.fq.gz with prefix:./MCC1.STAR STAR –runMode alignReads –chimSegmentMin 10 –genomeDir ./GenomeIndex_ENSEMBL_STAR_ReadLength49 –readFilesIn ./MCC1_1_trimmed.fq.gz –outFileNamePrefix ./MCC1.STAR. –alignEndsType Local –outSAMtype BAM SortedByCoordinate –outFilterMultimapNmax 20 –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04 –alignIntronMin 70 –alignIntronMax 1000000 –alignMatesGapMax 1000000 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –outSAMstrandField intronMotif –outFilterType BySJout –twopassMode Basic –outSAMmapqUnique 60 –limitGenomeGenerateRAM 208188325888 –runThreadN 128 –readFilesCommand zcat STAR version: 2.7.10b compiled: 2022-11-01T09:53:26-04:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 07 15:12:35 ….. started STAR run Mar 07 15:12:35 ….. loading genome Mar 07 15:13:13 ….. started 1st pass mapping Mar 07 15:13:36 ….. finished 1st pass mapping Mar 07 15:13:36 ….. inserting junctions into the genome indices Mar 07 15:14:22 ….. started mapping Mar 07 15:14:47 ….. finished mapping Mar 07 15:14:49 ….. started sorting BAM Mar 07 15:15:46 ….. finished successfully

[2023年 3月 7日 火曜日 15:15:49 JST] Indexes ./MCC1.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:15:51 JST] Converting ./MCC1.STAR.Aligned.sortedByCoord.out.bam to junc

[2023年 3月 7日 火曜日 15:16:12 JST] The Library Strand Type is: 0

[2023年 3月 7日 火曜日 15:16:12 JST] Run featureCounts to ./MCC1.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:16:16 JST] Please Check Read file: ./MCC2_1.fastq.gz

[2023年 3月 7日 火曜日 15:16:16 JST] Please Check Output Folder:./MCC2 The files ./MCC2_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 15:16:16 JST] Run trim_galore pigz 2.6

[2023年 3月 7日 火曜日 15:16:48 JST] FastQC of trimmed fastq files ./fastqc_results_trimmed/./MCC2_1_trimmed_fastqc.html Analysis complete for MCC2_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:17:43 JST] Using Salmon to Map sample 2 with 128 CPU cores: ./MCC2_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:18:29 JST] Mapping sample 2 with 128 CPU cores: ./MCC2_1_trimmed.fq.gz with prefix:./MCC2.STAR STAR –runMode alignReads –chimSegmentMin 10 –genomeDir ./GenomeIndex_ENSEMBL_STAR_ReadLength49 –readFilesIn ./MCC2_1_trimmed.fq.gz –outFileNamePrefix ./MCC2.STAR. –alignEndsType Local –outSAMtype BAM SortedByCoordinate –outFilterMultimapNmax 20 –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04 –alignIntronMin 70 –alignIntronMax 1000000 –alignMatesGapMax 1000000 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –outSAMstrandField intronMotif –outFilterType BySJout –twopassMode Basic –outSAMmapqUnique 60 –limitGenomeGenerateRAM 208188325888 –runThreadN 128 –readFilesCommand zcat STAR version: 2.7.10b compiled: 2022-11-01T09:53:26-04:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 07 15:18:29 ….. started STAR run Mar 07 15:18:29 ….. loading genome Mar 07 15:18:36 ….. started 1st pass mapping Mar 07 15:18:59 ….. finished 1st pass mapping Mar 07 15:19:00 ….. inserting junctions into the genome indices Mar 07 15:19:46 ….. started mapping Mar 07 15:20:11 ….. finished mapping Mar 07 15:20:13 ….. started sorting BAM Mar 07 15:21:13 ….. finished successfully

[2023年 3月 7日 火曜日 15:21:15 JST] Indexes ./MCC2.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:21:18 JST] Converting ./MCC2.STAR.Aligned.sortedByCoord.out.bam to junc

[2023年 3月 7日 火曜日 15:21:40 JST] The Library Strand Type is: 0

[2023年 3月 7日 火曜日 15:21:40 JST] Run featureCounts to ./MCC2.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:21:43 JST] Please Check Read file: ./MCC3_1.fastq.gz

[2023年 3月 7日 火曜日 15:21:43 JST] Please Check Output Folder:./MCC3 The files ./MCC3_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 15:21:43 JST] Run trim_galore pigz 2.6

[2023年 3月 7日 火曜日 15:22:15 JST] FastQC of trimmed fastq files ./fastqc_results_trimmed/./MCC3_1_trimmed_fastqc.html Analysis complete for MCC3_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:23:10 JST] Using Salmon to Map sample 3 with 128 CPU cores: ./MCC3_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:23:58 JST] Mapping sample 3 with 128 CPU cores: ./MCC3_1_trimmed.fq.gz with prefix:./MCC3.STAR STAR –runMode alignReads –chimSegmentMin 10 –genomeDir ./GenomeIndex_ENSEMBL_STAR_ReadLength49 –readFilesIn ./MCC3_1_trimmed.fq.gz –outFileNamePrefix ./MCC3.STAR. –alignEndsType Local –outSAMtype BAM SortedByCoordinate –outFilterMultimapNmax 20 –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04 –alignIntronMin 70 –alignIntronMax 1000000 –alignMatesGapMax 1000000 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –outSAMstrandField intronMotif –outFilterType BySJout –twopassMode Basic –outSAMmapqUnique 60 –limitGenomeGenerateRAM 208188325888 –runThreadN 128 –readFilesCommand zcat STAR version: 2.7.10b compiled: 2022-11-01T09:53:26-04:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 07 15:23:58 ….. started STAR run Mar 07 15:23:58 ….. loading genome Mar 07 15:24:06 ….. started 1st pass mapping Mar 07 15:24:29 ….. finished 1st pass mapping Mar 07 15:24:30 ….. inserting junctions into the genome indices Mar 07 15:25:16 ….. started mapping Mar 07 15:25:41 ….. finished mapping Mar 07 15:25:43 ….. started sorting BAM Mar 07 15:26:40 ….. finished successfully

[2023年 3月 7日 火曜日 15:26:43 JST] Indexes ./MCC3.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:26:45 JST] Converting ./MCC3.STAR.Aligned.sortedByCoord.out.bam to junc

[2023年 3月 7日 火曜日 15:27:07 JST] The Library Strand Type is: 0

[2023年 3月 7日 火曜日 15:27:07 JST] Run featureCounts to ./MCC3.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:27:10 JST] Please Check Read file: ./MCK1_1.fastq.gz

[2023年 3月 7日 火曜日 15:27:10 JST] Please Check Output Folder:./MCK1 The files ./MCK1_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 15:27:10 JST] Run trim_galore pigz 2.6

[2023年 3月 7日 火曜日 15:27:42 JST] FastQC of trimmed fastq files ./fastqc_results_trimmed/./MCK1_1_trimmed_fastqc.html Analysis complete for MCK1_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:28:36 JST] Using Salmon to Map sample 4 with 128 CPU cores: ./MCK1_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:29:32 JST] Mapping sample 4 with 128 CPU cores: ./MCK1_1_trimmed.fq.gz with prefix:./MCK1.STAR STAR –runMode alignReads –chimSegmentMin 10 –genomeDir ./GenomeIndex_ENSEMBL_STAR_ReadLength49 –readFilesIn ./MCK1_1_trimmed.fq.gz –outFileNamePrefix ./MCK1.STAR. –alignEndsType Local –outSAMtype BAM SortedByCoordinate –outFilterMultimapNmax 20 –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04 –alignIntronMin 70 –alignIntronMax 1000000 –alignMatesGapMax 1000000 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –outSAMstrandField intronMotif –outFilterType BySJout –twopassMode Basic –outSAMmapqUnique 60 –limitGenomeGenerateRAM 208188325888 –runThreadN 128 –readFilesCommand zcat STAR version: 2.7.10b compiled: 2022-11-01T09:53:26-04:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 07 15:29:32 ….. started STAR run Mar 07 15:29:32 ….. loading genome Mar 07 15:29:39 ….. started 1st pass mapping Mar 07 15:30:02 ….. finished 1st pass mapping Mar 07 15:30:02 ….. inserting junctions into the genome indices Mar 07 15:30:49 ….. started mapping Mar 07 15:31:13 ….. finished mapping Mar 07 15:31:14 ….. started sorting BAM Mar 07 15:32:16 ….. finished successfully

[2023年 3月 7日 火曜日 15:32:18 JST] Indexes ./MCK1.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:32:21 JST] Converting ./MCK1.STAR.Aligned.sortedByCoord.out.bam to junc

[2023年 3月 7日 火曜日 15:32:43 JST] The Library Strand Type is: 0

[2023年 3月 7日 火曜日 15:32:43 JST] Run featureCounts to ./MCK1.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:32:47 JST] Please Check Read file: ./MCK2_1.fastq.gz

[2023年 3月 7日 火曜日 15:32:47 JST] Please Check Output Folder:./MCK2 The files ./MCK2_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 15:32:47 JST] Run trim_galore pigz 2.6

[2023年 3月 7日 火曜日 15:33:19 JST] FastQC of trimmed fastq files ./fastqc_results_trimmed/./MCK2_1_trimmed_fastqc.html Analysis complete for MCK2_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:34:13 JST] Using Salmon to Map sample 5 with 128 CPU cores: ./MCK2_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:35:03 JST] Mapping sample 5 with 128 CPU cores: ./MCK2_1_trimmed.fq.gz with prefix:./MCK2.STAR STAR –runMode alignReads –chimSegmentMin 10 –genomeDir ./GenomeIndex_ENSEMBL_STAR_ReadLength49 –readFilesIn ./MCK2_1_trimmed.fq.gz –outFileNamePrefix ./MCK2.STAR. –alignEndsType Local –outSAMtype BAM SortedByCoordinate –outFilterMultimapNmax 20 –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04 –alignIntronMin 70 –alignIntronMax 1000000 –alignMatesGapMax 1000000 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –outSAMstrandField intronMotif –outFilterType BySJout –twopassMode Basic –outSAMmapqUnique 60 –limitGenomeGenerateRAM 208188325888 –runThreadN 128 –readFilesCommand zcat STAR version: 2.7.10b compiled: 2022-11-01T09:53:26-04:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 07 15:35:03 ….. started STAR run Mar 07 15:35:03 ….. loading genome Mar 07 15:35:10 ….. started 1st pass mapping Mar 07 15:35:34 ….. finished 1st pass mapping Mar 07 15:35:34 ….. inserting junctions into the genome indices Mar 07 15:36:20 ….. started mapping Mar 07 15:36:46 ….. finished mapping Mar 07 15:36:47 ….. started sorting BAM Mar 07 15:37:51 ….. finished successfully

[2023年 3月 7日 火曜日 15:37:53 JST] Indexes ./MCK2.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:37:56 JST] Converting ./MCK2.STAR.Aligned.sortedByCoord.out.bam to junc

[2023年 3月 7日 火曜日 15:38:18 JST] The Library Strand Type is: 0

[2023年 3月 7日 火曜日 15:38:18 JST] Run featureCounts to ./MCK2.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:38:22 JST] Please Check Read file: ./MCK3_1.fastq.gz

[2023年 3月 7日 火曜日 15:38:22 JST] Please Check Output Folder:./MCK3 The files ./MCK3_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 15:38:22 JST] Run trim_galore pigz 2.6

[2023年 3月 7日 火曜日 15:38:53 JST] FastQC of trimmed fastq files ./fastqc_results_trimmed/./MCK3_1_trimmed_fastqc.html Analysis complete for MCK3_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:39:48 JST] Using Salmon to Map sample 6 with 128 CPU cores: ./MCK3_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 15:40:33 JST] Mapping sample 6 with 128 CPU cores: ./MCK3_1_trimmed.fq.gz with prefix:./MCK3.STAR STAR –runMode alignReads –chimSegmentMin 10 –genomeDir ./GenomeIndex_ENSEMBL_STAR_ReadLength49 –readFilesIn ./MCK3_1_trimmed.fq.gz –outFileNamePrefix ./MCK3.STAR. –alignEndsType Local –outSAMtype BAM SortedByCoordinate –outFilterMultimapNmax 20 –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04 –alignIntronMin 70 –alignIntronMax 1000000 –alignMatesGapMax 1000000 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –outSAMstrandField intronMotif –outFilterType BySJout –twopassMode Basic –outSAMmapqUnique 60 –limitGenomeGenerateRAM 208188325888 –runThreadN 128 –readFilesCommand zcat STAR version: 2.7.10b compiled: 2022-11-01T09:53:26-04:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 07 15:40:33 ….. started STAR run Mar 07 15:40:33 ….. loading genome Mar 07 15:40:40 ….. started 1st pass mapping Mar 07 15:41:03 ….. finished 1st pass mapping Mar 07 15:41:04 ….. inserting junctions into the genome indices Mar 07 15:41:50 ….. started mapping Mar 07 15:42:15 ….. finished mapping Mar 07 15:42:17 ….. started sorting BAM Mar 07 15:43:24 ….. finished successfully

[2023年 3月 7日 火曜日 15:43:27 JST] Indexes ./MCK3.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:43:29 JST] Converting ./MCK3.STAR.Aligned.sortedByCoord.out.bam to junc

[2023年 3月 7日 火曜日 15:43:51 JST] The Library Strand Type is: 0

[2023年 3月 7日 火曜日 15:43:51 JST] Run featureCounts to ./MCK3.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:43:55 JST] FastQC of STAR bam files Analysis complete for MCC1.STAR.Aligned.sortedByCoord.out.bam Analysis complete for MCC3.STAR.Aligned.sortedByCoord.out.bam Analysis complete for MCC2.STAR.Aligned.sortedByCoord.out.bam Analysis complete for MCK3.STAR.Aligned.sortedByCoord.out.bam Analysis complete for MCK2.STAR.Aligned.sortedByCoord.out.bam Analysis complete for MCK1.STAR.Aligned.sortedByCoord.out.bam

[2023年 3月 7日 火曜日 15:45:18 JST] Create txt files that will be used to pass the group of inputs to LeafCutter.

[2023年 3月 7日 火曜日 15:45:18 JST] Run LeafCutter on 01.shVgll3vsControl.

[2023年 3月 7日 火曜日 15:45:18 JST] Run LeafCutter STEP1.

[2023年 3月 7日 火曜日 15:45:25 JST] Run LeafCutter STEP2. Loading counts from ./LeafCutter.Output/01.shVgll3vsControl_perind_numers.counts.gz Loading metadata from ./01.shVgll3vsControl.LeafCutter_groups.txt Encoding as shVgll3 =0, Control =1 Settings: $output_prefix [1] “./LeafCutter.Output/01.shVgll3vsControl”

$max_cluster_size [1] Inf

$min_samples_per_intron [1] 2

$min_samples_per_group [1] 2

$min_coverage [1] 20

$timeout [1] 30

$num_threads [1] 128

$exon_file [1] “./genome_filtered_exons.txt”

$init [1] “smart”

$seed [1] 12345

$help [1] FALSE

Running differential splicing analysis… Differential splicing summary: statuses Freq 1 <=1 sample with coverage>0 30 2 <=1 sample with coverage>min_coverage 2577 3 <2 introns used in >=min_samples_per_intron samples 665 4 Not enough valid samples 1015 5 Success 4489 Saving results… Loading exons from ./genome_filtered_exons.txt All done, exiting

[2023年 3月 7日 火曜日 15:45:52 JST] Run LeafCutter STEP3. Preparing for visualisation Results to be saved in: ./LeafCutter.Output/01.shVgll3vsControl.LeafCutter.RData Using annotation at: ./LeafCutter.Annotation/ Loading counts from ./LeafCutter.Output/01.shVgll3vsControl_perind_numers.counts.gz Loading metadata from ./01.shVgll3vsControl.LeafCutter_groups.txt Loading exons from ./LeafCutter.Annotation/_all_exons.txt.gz [1] “Annotating junctions” [1] “Preparing results” [1] “converting counts to ratios” [1] “creating PCA”

[2023年 3月 7日 火曜日 15:46:12 JST] Did not find bam list file, now create it

[2023年 3月 7日 火曜日 15:46:12 JST] This folder has 6 BAM files. |—-Now start transcript assemblies using stringtie [2023年 3月 7日 火曜日 15:46:13 JST] Transcript assemblies on BAM file No.1: ./MCC1.STAR.Aligned.sortedByCoord.out.bam.

[2023年 3月 7日 火曜日 15:47:04 JST] Transcript assemblies on BAM file No.2: ./MCC2.STAR.Aligned.sortedByCoord.out.bam.

[2023年 3月 7日 火曜日 15:47:54 JST] Transcript assemblies on BAM file No.3: ./MCC3.STAR.Aligned.sortedByCoord.out.bam.

[2023年 3月 7日 火曜日 15:48:44 JST] Transcript assemblies on BAM file No.4: ./MCK1.STAR.Aligned.sortedByCoord.out.bam.

[2023年 3月 7日 火曜日 15:49:35 JST] Transcript assemblies on BAM file No.5: ./MCK2.STAR.Aligned.sortedByCoord.out.bam.

[2023年 3月 7日 火曜日 15:50:25 JST] Transcript assemblies on BAM file No.6: ./MCK3.STAR.Aligned.sortedByCoord.out.bam.

[2023年 3月 7日 火曜日 15:51:16 JST] Merging transcript assemblies using StringTie.

[2023年 3月 7日 火曜日 15:51:29 JST] Remove Unstranded Transcripts.

[2023年 3月 7日 火曜日 15:51:32 JST] SQANTI3 QC. Rscript (R) version 4.2.2 (2022-10-31) List of 1 $ axis.text.x:List of 11 ..$ family : NULL ..$ face : NULL ..$ colour : NULL ..$ size : NULL ..$ hjust : num 1 ..$ vjust : NULL ..$ angle : num 60 ..$ lineheight : NULL ..$ margin : NULL ..$ debug : NULL ..$ inherit.blank: logi FALSE ..- attr(, “class”)= chr [1:2] “element_text” “element” - attr(, “class”)= chr [1:2] “theme” “gg” - attr(, “complete”)= logi FALSE - attr(, “validate”)= logi TRUE

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/home/mbb-wzy-ubuntu2/miniconda3/envs/SQANTI3.env/bin/pandoc +RTS -K512m -RTS /mnt/disk2/Yuan_Vgl3KO_ResOut/SQANTI3_report.knit.md –to html4 –from markdown+autolink_bare_uris+tex_math_single_backslash –output /mnt/disk2/Yuan_Vgl3KO_ResOut/temp_SQANTI3_report.html –lua-filter /home/mbb-wzy-ubuntu2/miniconda3/envs/SQANTI3.env/lib/R/library/rmarkdown/rmarkdown/lua/pagebreak.lua –lua-filter /home/mbb-wzy-ubuntu2/miniconda3/envs/SQANTI3.env/lib/R/library/rmarkdown/rmarkdown/lua/latex-div.lua –embed-resources –standalone –variable bs3=TRUE –section-divs –table-of-contents –toc-depth 3 –variable toc_float=1 –variable toc_selectors=h1,h2,h3 –variable toc_collapsed=1 –variable toc_smooth_scroll=1 –variable toc_print=1 –template /home/mbb-wzy-ubuntu2/miniconda3/envs/SQANTI3.env/lib/R/library/rmarkdown/rmd/h/default.html –no-highlight –variable highlightjs=1 –variable theme=bootstrap –css style.css –mathjax –variable ‘mathjax-url=https://mathjax.rstudio.com/latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML’ –include-in-header /tmp/RtmpL531XO/rmarkdown-str1d27bb28ff1467.html Write arguments to /mnt/disk2/Yuan_Vgl3KO_ResOut/temp.params.txt… **** Running SQANTI3… **** Parsing provided files…. Reading genome fasta /mnt/disk2/Yuan_Vgl3KO_ResOut/genome.fa…. Skipping aligning of sequences because GTF file was provided.

Indels will be not calculated since you ran SQANTI3 without alignment step (SQANTI3 with gtf format as transcriptome input). **** Predicting ORF sequences… **** Parsing Reference Transcriptome…. Splice Junction Coverage files not provided. **** TSS ratio will not be calculated since SR information was not provided **** Performing Classification of Isoforms…. Number of classified isoforms: 163049

[2023年 3月 7日 火曜日 16:01:47 JST] Make GenePred flat file for CIRCexplorer2.

[2023年 3月 7日 火曜日 16:01:52 JST] Extract transcript sequences using StringTie merged GTF.

[2023年 3月 7日 火曜日 16:01:58 JST] Re-Building salmon decoy with StringTie merged GTF **************** *** getDecoy ************* -j = 128 -a = /mnt/disk2/Yuan_Vgl3KO_ResOut/stdout_final.gtf -g = /mnt/disk2/Yuan_Vgl3KO_ResOut/genome.fa -t = /mnt/disk2/Yuan_Vgl3KO_ResOut/genomeTx_stdout.fa -o

= decoys [1/10] Extracting exonic features from the gtf [2/10] Masking the genome fasta [3/10] Aligning transcriptome to genome >>>>>>>>>>>>>>>>>> Reference = [reference.masked.genome.fa] Query = [/mnt/disk2/Yuan_Vgl3KO_ResOut/genomeTx_stdout.fa] Kmer size = 16 Window size = 5 Segment length = 500 (read split allowed) Alphabet = DNA Percentage identity threshold = 80% Mapping output file = mashmap.out Filter mode = 1 (1 = map, 2 = one-to-one, 3 = none) Execution threads = 128 >>>>>>>>>>>>>>>>>> INFO, skch::Sketch::build, minimizers picked from reference = 841883034 INFO, skch::Sketch::index, unique minimizers = 276208488 INFO, skch::Sketch::computeFreqHist, Frequency histogram of minimizers = (1, 141543152) … (2604270, 1) INFO, skch::Sketch::computeFreqHist, With threshold 0.001%, ignore minimizers occurring >= 7375 times during lookup. INFO, skch::main, Time spent computing the reference index: 227.8 sec INFO, skch::Map::mapQuery, [count of mapped reads, reads qualified for mapping, total input reads] = [135077, 135246, 163049] INFO, skch::main, Time spent mapping the query : 532.892 sec INFO, skch::main, mapping results saved in : mashmap.out [4/10] Extracting intervals from mashmap alignments [5/10] Merging the intervals [6/10] Extracting sequences from the genome [7/10] Concatenating to get decoy sequences [8/10] Making gentrome [9/10] Extracting decoy sequence ids [10/10] Removing temporary files

[2023年 3月 7日 火曜日 16:25:45 JST] Re-Building salmon index with StringTie merged GTF Threads = 128 Vertex length = 31 Hash functions = 5 Filter size = 8589934592 Capacity = 2 Files: salmon_index/ref_k31_fixed.fa ——————————————————————————– Round 0, 0:8589934592 Pass Filling Filtering 1 18 12
2 13 1 True junctions count = 1274503 False junctions count = 221362 Hash table size = 1495865 Candidate marks count = 14037012 ——————————————————————————– Reallocating bifurcations time: 1 True marks count: 13850228 Edges construction time: 14 ——————————————————————————– Distinct junctions = 1274503

for info, total work write each : 2.331 total work inram from level 3 : 4.322 total work raw : 25.000 Bitarray 1355924992 bits (100.00 %) (array + ranks ) final hash 0 bits (0.00 %) (nb in final hash 0)

[2023年 3月 7日 火曜日 16:27:31 JST] Please Check Read file: ./MCC1_1.fastq.gz

[2023年 3月 7日 火曜日 16:27:31 JST] Please Check Output Folder:./MCC1 The files ./MCC1_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 16:27:31 JST] Using Salmon to Map sample 1 with 128 CPU cores: ./MCC1_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 16:28:38 JST] Run CIRCexplorer2 on ./MCC1.STAR.Chimeric.out.junction

[2023年 3月 7日 火曜日 16:28:43 JST] Existing count file from featureCounts for ./MCC1.STAR.Aligned.sortedByCoord.out.bam, process to the next

[2023年 3月 7日 火曜日 16:28:43 JST] Please Check Read file: ./MCC2_1.fastq.gz

[2023年 3月 7日 火曜日 16:28:43 JST] Please Check Output Folder:./MCC2 The files ./MCC2_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 16:28:43 JST] Using Salmon to Map sample 2 with 128 CPU cores: ./MCC2_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 16:29:41 JST] Run CIRCexplorer2 on ./MCC2.STAR.Chimeric.out.junction

[2023年 3月 7日 火曜日 16:29:46 JST] Existing count file from featureCounts for ./MCC2.STAR.Aligned.sortedByCoord.out.bam, process to the next

[2023年 3月 7日 火曜日 16:29:46 JST] Please Check Read file: ./MCC3_1.fastq.gz

[2023年 3月 7日 火曜日 16:29:46 JST] Please Check Output Folder:./MCC3 The files ./MCC3_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 16:29:46 JST] Using Salmon to Map sample 3 with 128 CPU cores: ./MCC3_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 16:30:56 JST] Run CIRCexplorer2 on ./MCC3.STAR.Chimeric.out.junction

[2023年 3月 7日 火曜日 16:31:00 JST] Existing count file from featureCounts for ./MCC3.STAR.Aligned.sortedByCoord.out.bam, process to the next

[2023年 3月 7日 火曜日 16:31:00 JST] Please Check Read file: ./MCK1_1.fastq.gz

[2023年 3月 7日 火曜日 16:31:00 JST] Please Check Output Folder:./MCK1 The files ./MCK1_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 16:31:00 JST] Using Salmon to Map sample 4 with 128 CPU cores: ./MCK1_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 16:32:10 JST] Run CIRCexplorer2 on ./MCK1.STAR.Chimeric.out.junction

[2023年 3月 7日 火曜日 16:32:14 JST] Existing count file from featureCounts for ./MCK1.STAR.Aligned.sortedByCoord.out.bam, process to the next

[2023年 3月 7日 火曜日 16:32:14 JST] Please Check Read file: ./MCK2_1.fastq.gz

[2023年 3月 7日 火曜日 16:32:14 JST] Please Check Output Folder:./MCK2 The files ./MCK2_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 16:32:14 JST] Using Salmon to Map sample 5 with 128 CPU cores: ./MCK2_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 16:33:24 JST] Run CIRCexplorer2 on ./MCK2.STAR.Chimeric.out.junction

[2023年 3月 7日 火曜日 16:33:28 JST] Existing count file from featureCounts for ./MCK2.STAR.Aligned.sortedByCoord.out.bam, process to the next

[2023年 3月 7日 火曜日 16:33:28 JST] Please Check Read file: ./MCK3_1.fastq.gz

[2023年 3月 7日 火曜日 16:33:28 JST] Please Check Output Folder:./MCK3 The files ./MCK3_1.fastq.gz are encoded with: –Phred33

[2023年 3月 7日 火曜日 16:33:28 JST] Using Salmon to Map sample 6 with 128 CPU cores: ./MCK3_1_trimmed.fq.gz

[2023年 3月 7日 火曜日 16:34:43 JST] Run CIRCexplorer2 on ./MCK3.STAR.Chimeric.out.junction

[2023年 3月 7日 火曜日 16:34:48 JST] Existing count file from featureCounts for ./MCK3.STAR.Aligned.sortedByCoord.out.bam, process to the next

[2023年 3月 7日 火曜日 16:34:51 JST] Fix StringTie gtf. [1] “Make design matrix” [1] “Create switchAnalyzeRlist”

[2023年 3月 7日 火曜日 16:35:20 JST] SQANTI3 QC again. Rscript (R) version 4.2.2 (2022-10-31) List of 1 $ axis.text.x:List of 11 ..$ family : NULL ..$ face : NULL ..$ colour : NULL ..$ size : NULL ..$ hjust : num 1 ..$ vjust : NULL ..$ angle : num 60 ..$ lineheight : NULL ..$ margin : NULL ..$ debug : NULL ..$ inherit.blank: logi FALSE ..- attr(, “class”)= chr [1:2] “element_text” “element” - attr(, “class”)= chr [1:2] “theme” “gg” - attr(, “complete”)= logi FALSE - attr(, “validate”)= logi TRUE

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/home/mbb-wzy-ubuntu2/miniconda3/envs/SQANTI3.env/bin/pandoc +RTS -K512m -RTS /mnt/disk2/Yuan_Vgl3KO_ResOut/SQANTI3_report.knit.md –to html4 –from markdown+autolink_bare_uris+tex_math_single_backslash –output /mnt/disk2/Yuan_Vgl3KO_ResOut/temp_SQANTI3_report.html –lua-filter /home/mbb-wzy-ubuntu2/miniconda3/envs/SQANTI3.env/lib/R/library/rmarkdown/rmarkdown/lua/pagebreak.lua –lua-filter /home/mbb-wzy-ubuntu2/miniconda3/envs/SQANTI3.env/lib/R/library/rmarkdown/rmarkdown/lua/latex-div.lua –embed-resources –standalone –variable bs3=TRUE –section-divs –table-of-contents –toc-depth 3 –variable toc_float=1 –variable toc_selectors=h1,h2,h3 –variable toc_collapsed=1 –variable toc_smooth_scroll=1 –variable toc_print=1 –template /home/mbb-wzy-ubuntu2/miniconda3/envs/SQANTI3.env/lib/R/library/rmarkdown/rmd/h/default.html –no-highlight –variable highlightjs=1 –variable theme=bootstrap –css style.css –mathjax –variable ‘mathjax-url=https://mathjax.rstudio.com/latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML’ –include-in-header /tmp/RtmpUg7KaG/rmarkdown-str1d65736bd1478a.html Write arguments to /mnt/disk2/Yuan_Vgl3KO_ResOut/temp.params.txt… **** Running SQANTI3… **** Parsing provided files…. Reading genome fasta /mnt/disk2/Yuan_Vgl3KO_ResOut/genome.fa…. **** Predicting ORF sequences… **** Parsing Reference Transcriptome…. /mnt/disk2/Yuan_Vgl3KO_ResOut/refAnnotation_temp.genePred already exists. Using it. Splice Junction Coverage files not provided. **** TSS ratio will not be calculated since SR information was not provided **** Performing Classification of Isoforms…. Number of classified isoforms: 163049

[2023年 3月 7日 火曜日 16:44:37 JST] Run multiqc

[2023年 3月 7日 火曜日 16:46:19 JST] Create Reports

[2023年 3月 7日 火曜日 16:46:19 JST] Counting exon for DEXseq software

[2023年 3月 7日 火曜日 16:46:19 JST] Count number of reads that overlap with each of the exon counting bins of MCC1.STAR.Aligned.sortedByCoord.out.bam for using DEXseq R software with the following parameters: -f bam -p no -a 10 -s no

[2023年 3月 7日 火曜日 16:46:19 JST] Count number of reads that overlap with each of the exon counting bins of MCC2.STAR.Aligned.sortedByCoord.out.bam for using DEXseq R software with the following parameters: -f bam -p no -a 10 -s no

[2023年 3月 7日 火曜日 16:46:19 JST] Count number of reads that overlap with each of the exon counting bins of MCC3.STAR.Aligned.sortedByCoord.out.bam for using DEXseq R software with the following parameters: -f bam -p no -a 10 -s no

[2023年 3月 7日 火曜日 16:46:19 JST] Count number of reads that overlap with each of the exon counting bins of MCK3.STAR.Aligned.sortedByCoord.out.bam for using DEXseq R software with the following parameters: -f bam -p no -a 10 -s no

[2023年 3月 7日 火曜日 16:46:19 JST] Count number of reads that overlap with each of the exon counting bins of MCK1.STAR.Aligned.sortedByCoord.out.bam for using DEXseq R software with the following parameters: -f bam -p no -a 10 -s no

[2023年 3月 7日 火曜日 16:46:19 JST] Count number of reads that overlap with each of the exon counting bins of MCK2.STAR.Aligned.sortedByCoord.out.bam for using DEXseq R software with the following parameters: -f bam -p no -a 10 -s no

[2023年 3月 7日 火曜日 16:56:11 JST] Successfully Completed!! /usr/bin/pandoc [1] “2023-03-07 16:56:31 JST” [1] “Parameter setting for running DESeq2” [1] “2023-03-07 16:56:48 JST” [1] “Input Salmon counting results and batch correction”

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|================ | 23%error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length>

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|================================= | 47%error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length>

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|=================================================== | 72%error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length> error calling combine function: <simpleError in rbind(deparse.level, …): invalid list argument: all variables should have the same length>

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|======================================================================| 100%Creating package in ./00.Resource/TxDb.Mmu.2023.03.07.GRCm39 Creating package in 00.Resource/BSgenome.Mmu.2023.03.07.GRCm39 Loading ‘1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/1.fa’ … DONE Loading ‘10’ sequence from FASTA file ‘00.Resource/seqs_srcdir/10.fa’ … DONE Loading ‘11’ sequence from FASTA file ‘00.Resource/seqs_srcdir/11.fa’ … DONE Loading ‘12’ sequence from FASTA file ‘00.Resource/seqs_srcdir/12.fa’ … DONE Loading ‘13’ sequence from FASTA file ‘00.Resource/seqs_srcdir/13.fa’ … DONE Loading ‘14’ sequence from FASTA file ‘00.Resource/seqs_srcdir/14.fa’ … DONE Loading ‘15’ sequence from FASTA file ‘00.Resource/seqs_srcdir/15.fa’ … DONE Loading ‘16’ sequence from FASTA file ‘00.Resource/seqs_srcdir/16.fa’ … DONE Loading ‘17’ sequence from FASTA file ‘00.Resource/seqs_srcdir/17.fa’ … DONE Loading ‘18’ sequence from FASTA file ‘00.Resource/seqs_srcdir/18.fa’ … DONE Loading ‘19’ sequence from FASTA file ‘00.Resource/seqs_srcdir/19.fa’ … DONE Loading ‘2’ sequence from FASTA file ‘00.Resource/seqs_srcdir/2.fa’ … DONE Loading ‘3’ sequence from FASTA file ‘00.Resource/seqs_srcdir/3.fa’ … DONE Loading ‘4’ sequence from FASTA file ‘00.Resource/seqs_srcdir/4.fa’ … DONE Loading ‘5’ sequence from FASTA file ‘00.Resource/seqs_srcdir/5.fa’ … DONE Loading ‘6’ sequence from FASTA file ‘00.Resource/seqs_srcdir/6.fa’ … DONE Loading ‘7’ sequence from FASTA file ‘00.Resource/seqs_srcdir/7.fa’ … DONE Loading ‘8’ sequence from FASTA file ‘00.Resource/seqs_srcdir/8.fa’ … DONE Loading ‘9’ sequence from FASTA file ‘00.Resource/seqs_srcdir/9.fa’ … DONE Loading ‘GL456210.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456210.1.fa’ … DONE Loading ‘GL456211.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456211.1.fa’ … DONE Loading ‘GL456212.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456212.1.fa’ … DONE Loading ‘GL456219.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456219.1.fa’ … DONE Loading ‘GL456221.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456221.1.fa’ … DONE Loading ‘GL456233.2’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456233.2.fa’ … DONE Loading ‘GL456239.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456239.1.fa’ … DONE Loading ‘GL456354.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456354.1.fa’ … DONE Loading ‘GL456359.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456359.1.fa’ … DONE Loading ‘GL456360.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456360.1.fa’ … DONE Loading ‘GL456366.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456366.1.fa’ … DONE Loading ‘GL456367.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456367.1.fa’ … DONE Loading ‘GL456368.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456368.1.fa’ … DONE Loading ‘GL456370.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456370.1.fa’ … DONE Loading ‘GL456372.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456372.1.fa’ … DONE Loading ‘GL456378.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456378.1.fa’ … DONE Loading ‘GL456379.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456379.1.fa’ … DONE Loading ‘GL456381.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456381.1.fa’ … DONE Loading ‘GL456382.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456382.1.fa’ … DONE Loading ‘GL456383.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456383.1.fa’ … DONE Loading ‘GL456385.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456385.1.fa’ … DONE Loading ‘GL456387.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456387.1.fa’ … DONE Loading ‘GL456389.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456389.1.fa’ … DONE Loading ‘GL456390.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456390.1.fa’ … DONE Loading ‘GL456392.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456392.1.fa’ … DONE Loading ‘GL456394.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456394.1.fa’ … DONE Loading ‘GL456396.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/GL456396.1.fa’ … DONE Loading ‘JH584295.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584295.1.fa’ … DONE Loading ‘JH584296.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584296.1.fa’ … DONE Loading ‘JH584297.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584297.1.fa’ … DONE Loading ‘JH584298.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584298.1.fa’ … DONE Loading ‘JH584299.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584299.1.fa’ … DONE Loading ‘JH584300.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584300.1.fa’ … DONE Loading ‘JH584301.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584301.1.fa’ … DONE Loading ‘JH584302.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584302.1.fa’ … DONE Loading ‘JH584303.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584303.1.fa’ … DONE Loading ‘JH584304.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/JH584304.1.fa’ … DONE Loading ‘MT’ sequence from FASTA file ‘00.Resource/seqs_srcdir/MT.fa’ … DONE Loading ‘MU069434.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/MU069434.1.fa’ … DONE Loading ‘MU069435.1’ sequence from FASTA file ‘00.Resource/seqs_srcdir/MU069435.1.fa’ … DONE Loading ‘X’ sequence from FASTA file ‘00.Resource/seqs_srcdir/X.fa’ … DONE Loading ‘Y’ sequence from FASTA file ‘00.Resource/seqs_srcdir/Y.fa’ … DONE Writing all sequences to ‘00.Resource/BSgenome.Mmu.2023.03.07.GRCm39/inst/extdata/single_sequences.2bit’ … DONE

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|======================================================================| 100% used (Mb) gc trigger (Mb) max used (Mb) Ncells 17975767 960.1 49940841 2667.2 49940841 2667.2 Vcells 47332713 361.2 426481462 3253.8 666196016 5082.7 [1] “2023-03-07 17:31:32 JST” [1] “Pre-processing for DESeq2” [1] “Loading Annotation.Rdata” [1] “Loading Parameters.Rdata” [1] “Loading STARFeatureRawData.Rdata”

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|======================================================================| 100% used (Mb) gc trigger (Mb) max used (Mb) Ncells 18026265 962.8 49940841 2667.2 49940841 2667.2 Vcells 47452832 362.1 272948136 2082.5 666196016 5082.7 [1] “2023-03-07 17:32:45 JST” [1] “Running IsoformSwitchAnalyeR” [1] “Loading Parameters.Rdata” [1] “Loading STARFeatureRawData.Rdata” [1] “Loading Annotation.Rdata”

[2023年 3月 7日 火曜日 17:35:30 JST] Running CPC2

[2023年 3月 7日 火曜日 17:35:34 JST] Successfully Completed External Tools!!

[2023年 3月 7日 火曜日 17:35:35 JST] Running SignalP

[2023年 3月 7日 火曜日 17:36:01 JST] Running pfam_scan.pl

[2023年 3月 7日 火曜日 17:48:07 JST] Successfully Completed External Tools!!

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|======================================================================| 100% used (Mb) gc trigger (Mb) max used (Mb) Ncells 18833782 1005.9 49940841 2667.2 49940841 2667.2 Vcells 120503654 919.4 218358509 1666.0 666196016 5082.7 [1] “2023-03-07 17:59:55 JST” [1] “Functional enrichment analysis” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” [1] “This error is fine.” used (Mb) gc trigger (Mb) max used (Mb) Ncells 19736967 1054.1 49940841 2667.2 49940841 2667.2 Vcells 152571406 1164.1 418041165 3189.5 666196016 5082.7 [1] “2023-03-07 18:12:01 JST” [1] “Differential exon usage analysis” [1] “Loading Parameters.Rdata” [1] “Loading STARFeatureRawData.Rdata” [1] “Loading aSwitchList.Rdata” [1] “Loading Annotation.Rdata” [1] “Run DEXseq on DEXseq_01_shVgll3vsControl.” [1] “Transcription Subsetting” [1] “Estimate Size Factors” [1] “Estimate Dispersions” [1] “Test for DEU” [1] “Estimate Exon Fold Changes” used (Mb) gc trigger (Mb) max used (Mb) Ncells 19871520 1061.3 49940841 2667.2 49940841 2667.2 Vcells 152635094 1164.6 418041165 3189.5 666196016 5082.7 [1] “2023-03-07 18:16:33 JST” [1] “Circular RNA analysis” /usr/bin/pandoc [1] “Loading Parameters.Rdata” [1] “Loading STARFeatureRawData.Rdata” [1] “Loading aSwitchList.Rdata” [1] “Loading Annotation.Rdata” [1] “Loading DESeq2_Results.Rdata” [1] “Prepare project folder” [1] “Get BSJ from CIRCexplorer2” transcripts.txt is empty or absent. The longest transcripts for all circRNAs will be analyzed[1] “28 circRNA was identified.” [1] “First find frequency of single exon circRNAs” [1] “Retrieve random back-spliced junctions” [1] “Background target sequences” [1] “Find motifs in the background target sequences” motifs.txt is empty or absent. Only ATtRACT motifs will be analyzed if availablemotifs.txt is empty or absent. Only ATtRACT motifs will be analyzed if availablemotifs.txt is empty or absent. Only ATtRACT motifs will be analyzed if available[1] “Generating HTML report”

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|======================================================================| 100%[1] “Getting differentiatly-expressed circRNAs” transcripts.txt is empty or absent. The longest transcripts for all circRNAs will be analyzed used (Mb) gc trigger (Mb) max used (Mb) Ncells 19934980 1064.7 49940841 2667.2 49940841 2667.2 Vcells 152776598 1165.6 418041165 3189.5 666196016 5082.7 [1] “2023-03-07 18:22:28 JST” [1] “Output website summary” /usr/bin/pandoc [1] “Loading Parameters.Rdata” [1] “Loading STARFeatureRawData.Rdata” [1] “Loading aSwitchList.Rdata” [1] “Loading Annotation.Rdata” [1] “Loading DESeq2_Results.Rdata” [1] “Loading DEXseqResOut.Rdata” [1] “Loading circRNA_Results.Rdata” [1] “Loading DESeq2_Processing.Rdata” [1] “Make webpage for shVgll3vsControl” [1] “Make webpage for each gene in shVgll3vsControl”

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|======================================================================| 100%Allowing parallel execution with up to 127 working processes. pickSoftThreshold: will use block size 2937. pickSoftThreshold: calculating connectivity for given powers… ..working on genes 1 through 2937 of 15232 ..working on genes 2938 through 5874 of 15232 ..working on genes 5875 through 8811 of 15232 ..working on genes 8812 through 11748 of 15232 ..working on genes 11749 through 14685 of 15232 ..working on genes 14686 through 15232 of 15232 Power SFT.R.sq slope truncated.R.sq mean.k. median.k. max.k. 1 1 0.5020 1.0400 0.726 7420 7810.0 9810 2 2 0.0153 0.0838 0.855 4820 4960.0 7670 3 3 0.1450 -0.2290 0.973 3530 3440.0 6430 4 4 0.3790 -0.3860 0.991 2750 2520.0 5600 5 5 0.5370 -0.4820 0.996 2240 1910.0 4990 6 6 0.6390 -0.5480 0.993 1880 1490.0 4520 7 7 0.7170 -0.5990 0.993 1610 1180.0 4140 8 8 0.7680 -0.6440 0.994 1400 962.0 3830 9 9 0.8110 -0.6750 0.992 1230 793.0 3560 10 10 0.8440 -0.7040 0.991 1100 661.0 3340 11 11 0.8650 -0.7260 0.988 985 558.0 3140 12 12 0.8810 -0.7490 0.988 892 475.0 2960 13 13 0.8940 -0.7670 0.987 813 410.0 2810 14 14 0.9080 -0.7850 0.987 745 356.0 2670 15 15 0.9190 -0.8000 0.986 686 312.0 2550 16 16 0.9270 -0.8130 0.986 635 275.0 2430 17 17 0.9350 -0.8270 0.986 590 245.0 2330 18 18 0.9410 -0.8380 0.986 549 219.0 2240 19 19 0.9450 -0.8490 0.986 514 197.0 2150 20 20 0.9520 -0.8620 0.986 482 178.0 2070 21 21 0.9560 -0.8730 0.985 453 161.0 1990 22 22 0.9590 -0.8820 0.984 427 147.0 1930 23 23 0.9610 -0.8890 0.984 403 135.0 1860 24 24 0.9650 -0.8980 0.985 381 124.0 1800 25 25 0.9680 -0.9060 0.984 361 114.0 1740 26 26 0.9700 -0.9130 0.985 343 106.0 1690 27 27 0.9720 -0.9200 0.984 326 98.2 1640 28 28 0.9730 -0.9260 0.985 311 91.3 1590 29 29 0.9760 -0.9340 0.985 297 85.1 1550 30 30 0.9780 -0.9410 0.985 283 79.4 1510 Calculating module eigengenes block-wise from all genes Flagging genes and samples with too many missing values… ..step 1 ….pre-clustering genes to determine blocks.. Projective K-means: ..using 101 centers. ..k-means clustering.. ..iteration 1 ..proposing to move 14987 genes..move accepted. ..iteration 2 ..proposing to move 7446 genes..move accepted. ..iteration 3 ..proposing to move 4821 genes..move accepted. ..iteration 4 ..proposing to move 3590 genes..move accepted. ..iteration 5 ..proposing to move 3072 genes..move accepted. ..iteration 6 ..proposing to move 2651 genes..move accepted. ..iteration 7 ..proposing to move 2277 genes..move accepted. ..iteration 8 ..proposing to move 1906 genes..move accepted. ..iteration 9 ..proposing to move 1650 genes..move accepted. ..iteration 10 ..proposing to move 1390 genes..move accepted. ..iteration 11 ..proposing to move 1216 genes..move accepted. ..iteration 12 ..proposing to move 1069 genes..move accepted. ..iteration 13 ..proposing to move 988 genes..move accepted. ..iteration 14 ..proposing to move 834 genes..move accepted. ..iteration 15 ..proposing to move 735 genes..move accepted. ..iteration 16 ..proposing to move 647 genes..move accepted. ..iteration 17 ..proposing to move 614 genes..move accepted. ..iteration 18 ..proposing to move 575 genes..move accepted. ..iteration 19 ..proposing to move 532 genes..move accepted. ..iteration 20 ..proposing to move 476 genes..move accepted. ..iteration 21 ..proposing to move 424 genes..move accepted. ..iteration 22 ..proposing to move 381 genes..move accepted. ..iteration 23 ..proposing to move 308 genes..move accepted. ..iteration 24 ..proposing to move 276 genes..move accepted. ..iteration 25 ..proposing to move 269 genes..move accepted. ..iteration 26 ..proposing to move 241 genes..move accepted. ..iteration 27 ..proposing to move 238 genes..move accepted. ..iteration 28 ..proposing to move 195 genes..move accepted. ..iteration 29 ..proposing to move 166 genes..move accepted. ..iteration 30 ..proposing to move 139 genes..move accepted. ..iteration 31 ..proposing to move 126 genes..move accepted. ..iteration 32 ..proposing to move 131 genes..move accepted. ..iteration 33 ..proposing to move 108 genes..move accepted. ..iteration 34 ..proposing to move 83 genes..move accepted. ..iteration 35 ..proposing to move 71 genes..move accepted. ..iteration 36 ..proposing to move 40 genes..move accepted. ..iteration 37 ..proposing to move 33 genes..move accepted. ..iteration 38 ..proposing to move 20 genes..move accepted. ..iteration 39 ..proposing to move 14 genes..move accepted. ..iteration 40 ..proposing to move 6 genes..move accepted. ..iteration 41 Could not find a suitable move to improve the clustering. Sizes of preliminary clusters: membership 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 145 337 130 275 100 113 165 177 114 117 126 197 184 125 114 161 107 128 235 120 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 108 107 114 161 130 107 115 94 111 212 285 88 128 101 131 103 108 120 153 109 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 125 141 96 593 125 131 149 110 117 93 115 120 133 215 126 129 205 159 370 108 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 127 85 115 139 177 268 99 273 152 192 187 290 161 108 168 105 113 118 129 349 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 121 148 133 138 177 183 169 215 141 439 139 133 89 128 130 129 150 ..merging smaller clusters… Block sizes: gBlocks 1 2 14246 986 ..Working on block 1 . TOM calculation: adjacency.. ..will use 127 parallel threads. Fraction of slow calculations: 0.000000 ..connectivity.. ..matrix multiplication (system BLAS).. ..normalization.. ..done. ..saving TOM for block 1 into file WGCNA_res-block.1.RData ….clustering.. ….detecting modules.. ..done. ….calculating module eigengenes.. moduleEigengenes : Working on ME for module 1 moduleEigengenes : Working on ME for module 2 moduleEigengenes : Working on ME for module 3 moduleEigengenes : Working on ME for module 4 moduleEigengenes : Working on ME for module 5 moduleEigengenes : Working on ME for module 6 moduleEigengenes : Working on ME for module 7 moduleEigengenes : Working on ME for module 8 moduleEigengenes : Working on ME for module 9 moduleEigengenes : Working on ME for module 10 moduleEigengenes : Working on ME for module 11 moduleEigengenes : Working on ME for module 12 moduleEigengenes : Working on ME for module 13 moduleEigengenes : Working on ME for module 14 moduleEigengenes : Working on ME for module 15 moduleEigengenes : Working on ME for module 16 moduleEigengenes : Working on ME for module 17 moduleEigengenes : Working on ME for module 18 moduleEigengenes : Working on ME for module 19 moduleEigengenes : Working on ME for module 20 moduleEigengenes : Working on ME for module 21 moduleEigengenes : Working on ME for module 22 moduleEigengenes : Working on ME for module 23 moduleEigengenes : Working on ME for module 24 moduleEigengenes : Working on ME for module 25 moduleEigengenes : Working on ME for module 26 moduleEigengenes : Working on ME for module 27 moduleEigengenes : Working on ME for module 28 moduleEigengenes : Working on ME for module 29 moduleEigengenes : Working on ME for module 30 moduleEigengenes : Working on ME for module 31 moduleEigengenes : Working on ME for module 32 moduleEigengenes : Working on ME for module 33 moduleEigengenes : Working on ME for module 34 moduleEigengenes : Working on ME for module 35 moduleEigengenes : Working on ME for module 36 moduleEigengenes : Working on ME for module 37 moduleEigengenes : Working on ME for module 38 moduleEigengenes : Working on ME for module 39 moduleEigengenes : Working on ME for module 40 moduleEigengenes : Working on ME for module 41 moduleEigengenes : Working on ME for module 42 moduleEigengenes : Working on ME for module 43 moduleEigengenes : Working on ME for module 44 moduleEigengenes : Working on ME for module 45 moduleEigengenes : Working on ME for module 46 moduleEigengenes : Working on ME for module 47 moduleEigengenes : Working on ME for module 48 moduleEigengenes : Working on ME for module 49 moduleEigengenes : Working on ME for module 50 moduleEigengenes : Working on ME for module 51 moduleEigengenes : Working on ME for module 52 moduleEigengenes : Working on ME for module 53 moduleEigengenes : Working on ME for module 54 moduleEigengenes : Working on ME for module 55 moduleEigengenes : Working on ME for module 56 moduleEigengenes : Working on ME for module 57 moduleEigengenes : Working on ME for module 58 moduleEigengenes : Working on ME for module 59 moduleEigengenes : Working on ME for module 60 moduleEigengenes : Working on ME for module 61 moduleEigengenes : Working on ME for module 62 moduleEigengenes : Working on ME for module 63 moduleEigengenes : Working on ME for module 64 moduleEigengenes : Working on ME for module 65 moduleEigengenes : Working on ME for module 66 moduleEigengenes : Working on ME for module 67 moduleEigengenes : Working on ME for module 68 moduleEigengenes : Working on ME for module 69 moduleEigengenes : Working on ME for module 70 moduleEigengenes : Working on ME for module 71 moduleEigengenes : Working on ME for module 72 moduleEigengenes : Working on ME for module 73 moduleEigengenes : Working on ME for module 74 moduleEigengenes : Working on ME for module 75 moduleEigengenes : Working on ME for module 76 moduleEigengenes : Working on ME for module 77 moduleEigengenes : Working on ME for module 78 moduleEigengenes : Working on ME for module 79 moduleEigengenes : Working on ME for module 80 moduleEigengenes : Working on ME for module 81 moduleEigengenes : Working on ME for module 82 moduleEigengenes : Working on ME for module 83 moduleEigengenes : Working on ME for module 84 moduleEigengenes : Working on ME for module 85 moduleEigengenes : Working on ME for module 86 moduleEigengenes : Working on ME for module 87 moduleEigengenes : Working on ME for module 88 moduleEigengenes : Working on ME for module 89 moduleEigengenes : Working on ME for module 90 moduleEigengenes : Working on ME for module 91 moduleEigengenes : Working on ME for module 92 moduleEigengenes : Working on ME for module 93 moduleEigengenes : Working on ME for module 94 moduleEigengenes : Working on ME for module 95 moduleEigengenes : Working on ME for module 96 moduleEigengenes : Working on ME for module 97 moduleEigengenes : Working on ME for module 98 moduleEigengenes : Working on ME for module 99 moduleEigengenes : Working on ME for module 100 moduleEigengenes : Working on ME for module 101 moduleEigengenes : Working on ME for module 102 moduleEigengenes : Working on ME for module 103 moduleEigengenes : Working on ME for module 104 moduleEigengenes : Working on ME for module 105 moduleEigengenes : Working on ME for module 106 moduleEigengenes : Working on ME for module 107 moduleEigengenes : Working on ME for module 108 moduleEigengenes : Working on ME for module 109 moduleEigengenes : Working on ME for module 110 moduleEigengenes : Working on ME for module 111 moduleEigengenes : Working on ME for module 112 moduleEigengenes : Working on ME for module 113 moduleEigengenes : Working on ME for module 114 moduleEigengenes : Working on ME for module 115 moduleEigengenes : Working on ME for module 116 moduleEigengenes : Working on ME for module 117 moduleEigengenes : Working on ME for module 118 moduleEigengenes : Working on ME for module 119 moduleEigengenes : Working on ME for module 120 moduleEigengenes : Working on ME for module 121 moduleEigengenes : Working on ME for module 122 moduleEigengenes : Working on ME for module 123 moduleEigengenes : Working on ME for module 124 moduleEigengenes : Working on ME for module 125 moduleEigengenes : Working on ME for module 126 moduleEigengenes : Working on ME for module 127 moduleEigengenes : Working on ME for module 128 moduleEigengenes : Working on ME for module 129 moduleEigengenes : Working on ME for module 130 moduleEigengenes : Working on ME for module 131 moduleEigengenes : Working on ME for module 132 moduleEigengenes : Working on ME for module 133 moduleEigengenes : Working on ME for module 134 moduleEigengenes : Working on ME for module 135 moduleEigengenes : Working on ME for module 136 moduleEigengenes : Working on ME for module 137 moduleEigengenes : Working on ME for module 138 moduleEigengenes : Working on ME for module 139 moduleEigengenes : Working on ME for module 140 moduleEigengenes : Working on ME for module 141 moduleEigengenes : Working on ME for module 142 moduleEigengenes : Working on ME for module 143 moduleEigengenes : Working on ME for module 144 moduleEigengenes : Working on ME for module 145 moduleEigengenes : Working on ME for module 146 moduleEigengenes : Working on ME for module 147 moduleEigengenes : Working on ME for module 148 moduleEigengenes : Working on ME for module 149 moduleEigengenes : Working on ME for module 150 moduleEigengenes : Working on ME for module 151 moduleEigengenes : Working on ME for module 152 moduleEigengenes : Working on ME for module 153 moduleEigengenes : Working on ME for module 154 moduleEigengenes : Working on ME for module 155 moduleEigengenes : Working on ME for module 156 moduleEigengenes : Working on ME for module 157 moduleEigengenes : Working on ME for module 158 moduleEigengenes : Working on ME for module 159 moduleEigengenes : Working on ME for module 160 moduleEigengenes : Working on ME for module 161 moduleEigengenes : Working on ME for module 162 moduleEigengenes : Working on ME for module 163 moduleEigengenes : Working on ME for module 164 moduleEigengenes : Working on ME for module 165 moduleEigengenes : Working on ME for module 166 moduleEigengenes : Working on ME for module 167 moduleEigengenes : Working on ME for module 168 ….checking kME in modules.. ..Working on block 2 . TOM calculation: adjacency.. ..will use 127 parallel threads. Fraction of slow calculations: 0.000000 ..connectivity.. ..matrix multiplication (system BLAS).. ..normalization.. ..done. ..saving TOM for block 2 into file WGCNA_res-block.2.RData ….clustering.. ….detecting modules.. ..done. ….calculating module eigengenes.. moduleEigengenes : Working on ME for module 169 moduleEigengenes : Working on ME for module 170 moduleEigengenes : Working on ME for module 171 moduleEigengenes : Working on ME for module 172 moduleEigengenes : Working on ME for module 173 moduleEigengenes : Working on ME for module 174 moduleEigengenes : Working on ME for module 175 moduleEigengenes : Working on ME for module 176 moduleEigengenes : Working on ME for module 177 moduleEigengenes : Working on ME for module 178 moduleEigengenes : Working on ME for module 179 moduleEigengenes : Working on ME for module 180 moduleEigengenes : Working on ME for module 181 moduleEigengenes : Working on ME for module 182 moduleEigengenes : Working on ME for module 183 moduleEigengenes : Working on ME for module 184 moduleEigengenes : Working on ME for module 185 moduleEigengenes : Working on ME for module 186 moduleEigengenes : Working on ME for module 187 ….checking kME in modules.. ..merging modules that are too close.. mergeCloseModules: Merging modules whose distance is less than 0.3 multiSetMEs: Calculating module MEs. Working on set 1 … moduleEigengenes: Calculating 187 module eigengenes in given set. multiSetMEs: Calculating module MEs. Working on set 1 … moduleEigengenes: Calculating 34 module eigengenes in given set. multiSetMEs: Calculating module MEs. Working on set 1 … moduleEigengenes: Calculating 22 module eigengenes in given set. multiSetMEs: Calculating module MEs. Working on set 1 … moduleEigengenes: Calculating 20 module eigengenes in given set. Calculating new MEs… multiSetMEs: Calculating module MEs. Working on set 1 … moduleEigengenes: Calculating 20 module eigengenes in given set.

[2023年 3月 7日 火曜日 18:32:51 JST] zip mapped BAM files adding: MCC1.STAR.Aligned.sortedByCoord.out.bam (deflated 0%) adding: MCC1.STAR.Aligned.sortedByCoord.out.bam.bai (deflated 70%) adding: MCC2.STAR.Aligned.sortedByCoord.out.bam (deflated 0%) adding: MCC2.STAR.Aligned.sortedByCoord.out.bam.bai (deflated 71%) adding: MCC3.STAR.Aligned.sortedByCoord.out.bam (deflated 0%) adding: MCC3.STAR.Aligned.sortedByCoord.out.bam.bai (deflated 70%) adding: MCK1.STAR.Aligned.sortedByCoord.out.bam (deflated 0%) adding: MCK1.STAR.Aligned.sortedByCoord.out.bam.bai (deflated 70%) adding: MCK2.STAR.Aligned.sortedByCoord.out.bam (deflated 0%) adding: MCK2.STAR.Aligned.sortedByCoord.out.bam.bai (deflated 71%) adding: MCK3.STAR.Aligned.sortedByCoord.out.bam (deflated 0%) adding: MCK3.STAR.Aligned.sortedByCoord.out.bam.bai (deflated 70%)

[2023年 3月 7日 火曜日 18:36:50 JST] zip de novo assembly transcriptome

[2023年 3月 7日 火曜日 18:36:50 JST] Zip LeafCutter results adding: LeafCutter.Output/ (stored 0%) adding: LeafCutter.Output/01.shVgll3vsControl_refined (deflated 67%) adding: LeafCutter.Output/01.shVgll3vsControl_cluster_significance.txt (deflated 73%) adding: LeafCutter.Output/01.shVgll3vsControl_perind.counts.gz (deflated 0%) adding: LeafCutter.Output/01.shVgll3vsControl_perind_numers.counts.gz (deflated 0%) adding: LeafCutter.Output/01.shVgll3vsControl.LeafCutter.RData (deflated 0%) adding: LeafCutter.Output/01.shVgll3vsControl_sortedlibs (deflated 75%) adding: LeafCutter.Output/01.shVgll3vsControl_pooled (deflated 66%) adding: LeafCutter.Output/01.shVgll3vsControl_effect_sizes.txt (deflated 62%)

[2023年 3月 7日 火曜日 18:36:55 JST] Zip circular RNA results adding: CircRNA_res/ (stored 0%) adding: CircRNA_res/MCK1.STAR.SJ.out.tab (deflated 70%) adding: CircRNA_res/MCC2.STAR.Chimeric.out.junction (deflated 70%) adding: CircRNA_res/MCK2.STAR.Chimeric.out.junction (deflated 71%) adding: CircRNA_res/MCC3.STAR.SJ.out.tab (deflated 70%) adding: CircRNA_res/MCC3_CIRCexplorer2.ce (deflated 66%) adding: CircRNA_res/MCC2_CIRCexplorer2.ce (deflated 69%) adding: CircRNA_res/MCK3.STAR.Chimeric.out.junction (deflated 71%) adding: CircRNA_res/MCC1.STAR.SJ.out.tab (deflated 70%) adding: CircRNA_res/MCK2_CIRCexplorer2.ce (deflated 64%) adding: CircRNA_res/MCK1.STAR.Chimeric.out.junction (deflated 70%) adding: CircRNA_res/MCK3_CIRCexplorer2.ce (deflated 65%) adding: CircRNA_res/MCC1_CIRCexplorer2.ce (deflated 66%) adding: CircRNA_res/MCK2.STAR.SJ.out.tab (deflated 70%) adding: CircRNA_res/MCC2.STAR.SJ.out.tab (deflated 70%) adding: CircRNA_res/MCK3.STAR.SJ.out.tab (deflated 70%) adding: CircRNA_res/MCK1_CIRCexplorer2.ce (deflated 66%) adding: CircRNA_res/MCC1.STAR.Chimeric.out.junction (deflated 71%) adding: CircRNA_res/MCC3.STAR.Chimeric.out.junction (deflated 71%)

[2023年 3月 7日 火曜日 18:36:57 JST] Zip BSgenome package adding: BSgenome.Mmu.2023.03.07.GRCm39/ (stored 0%) adding: BSgenome.Mmu.2023.03.07.GRCm39/man/ (stored 0%) adding: BSgenome.Mmu.2023.03.07.GRCm39/man/package.Rd (deflated 62%) adding: BSgenome.Mmu.2023.03.07.GRCm39/R/ (stored 0%) adding: BSgenome.Mmu.2023.03.07.GRCm39/R/zzz.R (deflated 62%) adding: BSgenome.Mmu.2023.03.07.GRCm39/DESCRIPTION (deflated 42%) adding: BSgenome.Mmu.2023.03.07.GRCm39/NAMESPACE (deflated 37%) adding: BSgenome.Mmu.2023.03.07.GRCm39/inst/ (stored 0%) adding: BSgenome.Mmu.2023.03.07.GRCm39/inst/extdata/ (stored 0%) adding: BSgenome.Mmu.2023.03.07.GRCm39/inst/extdata/single_sequences.2bit (deflated 8%)

[2023年 3月 7日 火曜日 18:37:22 JST] Zip TxDb package adding: TxDb.Mmu.2023.03.07.GRCm39/ (stored 0%) adding: TxDb.Mmu.2023.03.07.GRCm39/man/ (stored 0%) adding: TxDb.Mmu.2023.03.07.GRCm39/man/package.Rd (deflated 51%) adding: TxDb.Mmu.2023.03.07.GRCm39/R/ (stored 0%) adding: TxDb.Mmu.2023.03.07.GRCm39/R/zzz.R (deflated 46%) adding: TxDb.Mmu.2023.03.07.GRCm39/DESCRIPTION (deflated 40%) adding: TxDb.Mmu.2023.03.07.GRCm39/NAMESPACE (deflated 21%) adding: TxDb.Mmu.2023.03.07.GRCm39/inst/ (stored 0%) adding: TxDb.Mmu.2023.03.07.GRCm39/inst/extdata/ (stored 0%) adding: TxDb.Mmu.2023.03.07.GRCm39/inst/extdata/TxDb.Mmu.2023.03.07.GRCm39.sqlite (deflated 61%)

[2023年 3月 7日 火曜日 18:37:29 JST] Successfully Zipped Downloadable Content!!

2021 - | 王 紫仪 (WANG Ziyi) |