Star2.7.9a twopassMode basic not properly aligning my reads nor giving me proper output sam/bam files

[tsimoneaux]

I am trying to run Star using the twopassmode basic option but, I am not getting output sam/bam files. I have copied my script. The onscreen message appears and says mapping and finished successfully after a couple of minutes. There is output of intermediate files but no sam or bam file outputs.

$STAR --twopassMode Basic --genomeDir /home/Tankya/Star/STAR_INDEX_GRCh38 --runThreadN 16 --readFilesCommand zcat --outReadsUnmapped Fastx --outFilterType BySJout --outSAMtype BAM SortedByCoordinate -- quantMode GeneCounts --outFileNamePrefix GRCh38 --readFilesIn $i --outSAMheaderHD @HD VN:1.4 SO:coordinate --outSAMattrRGline ID:4 lb:lib1 PL:iontorrent SM:$i PU:$i

[alexdobin]

Hi @tsimoneaux

this could be an issue with unzipping the files. Please try to run it on uncompressed FASTQ without --readFilesCommand zcat

Cheers Alex

[tsimoneaux]

Thanks Alex! Also can STAR align without an annotation file? I have a pangenome I am trying to use as my reference and it doesn't have an annotation file.

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From: Alexander Dobin @.> Sent: Saturday, August 14, 2021 12:32 PM To: alexdobin/STAR @.> Cc: Simoneaux, Tankya @.>; Mention @.> Subject: Re: [alexdobin/STAR] Star2.7.9a twopassMode basic not properly aligning my reads nor giving me proper output sam/bam files (#1317)

CAUTION! This email came from outside of Morehouse School of Medicine. Exercise extra caution clicking links and opening attachments from any and all senders.

Hi @tsimoneauxhttps://github.com/tsimoneaux

this could be an issue with unzipping the files. Please try to run it on uncompressed FASTQ without --readFilesCommand zcat

Cheers Alex

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/alexdobin/STAR/issues/1317#issuecomment-898915634, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AHHEWH2RI2ADO4CZXGYVTHDT42LCTANCNFSM5BRZV4OQ. Triage notifications on the go with GitHub Mobile for iOShttps://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675 or Androidhttps://play.google.com/store/apps/details?id=com.github.android&utm_campaign=notification-email.

[alexdobin]

Hi @tsimoneaux

you can run STAR without annotations, but then you cannot use --quantMode GeneCounts option, which requires knowledge of gene/transcript coordinates.

Cheers Alex

[tsimoneaux]

Hi Alex need serious help troubleshooting. I am trying to align some RNAseq data but the output files are empty. Here is what I see inside the Log.out file

Mar 17 18:56:14 ..... started STAR run Mar 17 18:56:14 ..... loading genome Mar 17 18:59:19 ..... processing annotations GTF Mar 17 18:59:47 ..... inserting junctions into the genome indices Mar 17 19:00:09 ..... started 1st pass mapping /DATA/PROJECTS/Stroke2/Scripts2/03A_GRCH38+APG.sh: line 72: 300288 Killed STAR --twopassMode Basic --limitBAMsortRAM 2000000000 --genomeDir /DATA/REFERENCES/AfricanPanG+GRCH38 --outReadsUnmapped Fastx --sjdbGTFfile /DATA/REFERENCES/GRCH38_MainChrs/Homo_sapiens.GRCh38.104.gtf --outFilterType BySJout --outSAMattributes NH HI AS nM NM MD jM jI MC ch --outSAMattrRGline ID:S0370_134_G12_R1_S5-00534_167_01 PL:ION-TORRENT PU:S0370_134_G12_R1_S5-00534_167_01 LB:S0370 SM:S0370 --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --readFilesCommand zcat --runThreadN 8 --chimOutType Junctions SeparateSAMold WithinBAM SoftClip --chimOutJunctionFormat 1 --chimSegmentMin 15 --limitOutSJcollapsed 5000000 --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM GeneCounts --outSAMheaderHD @HD VN:1.4 SO:coordinate --outFileNamePrefix /DATA/PROJECTS/Stroke2/ANALYSIS/S0370/03A_APG+GRCH38_AlignedData/S0370_134_G12_R1_S5-00534_167_01/S0370_134_G12_R1_S5-00534_167_01 --readFilesIn /DATA/PROJECTS/Stroke2/ANALYSIS/S0370/02_TrimmedFastq/S0370_134_G12_R1_S5-00534_167_01_trimmed.fq.gz /DATA/PROJECTS/Stroke2/Scripts2/03A_GRCH38+APG.sh: line 72: 300385 Killed STAR --twopassMode Basic --limitBAMsortRAM 2000000000 --genomeDir /DATA/REFERENCES/AfricanPanG+GRCH38 --outReadsUnmapped Fastx --sjdbGTFfile /DATA/REFERENCES/GRCH38_MainChrs/Homo_sapiens.GRCh38.104.gtf --outFilterType BySJout --outSAMattributes NH HI AS nM NM MD jM jI MC ch --outSAMattrRGline ID:S0370_135_G12_R2_S5-00534_168_01 PL:ION-TORRENT PU:S0370_135_G12_R2_S5-00534_168_01 LB:S0370 SM:S0370 --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --readFilesCommand zcat --runThreadN 8 --chimOutType Junctions SeparateSAMold WithinBAM SoftClip --chimOutJunctionFormat 1 --chimSegmentMin 15 --limitOutSJcollapsed 5000000 --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM GeneCounts --outSAMheaderHD @HD VN:1.4 SO:coordinate --outFileNamePrefix /DATA/PROJECTS/Stroke2/ANALYSIS/S0370/03A_APG+GRCH38_AlignedData/S0370_135_G12_R2_S5-00534_168_01/S0370_135_G12_R2_S5-00534_168_01 --readFilesIn /DATA/PROJECTS/Stroke2/ANALYSIS/S0370/02_TrimmedFastq/S0370_135_G12_R2_S5-00534_168_01_trimmed.fq.gz

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From: Alexander Dobin @.> Sent: Tuesday, August 17, 2021 5:48 PM To: alexdobin/STAR @.> Cc: Simoneaux, Tankya @.>; Mention @.> Subject: Re: [alexdobin/STAR] Star2.7.9a twopassMode basic not properly aligning my reads nor giving me proper output sam/bam files (#1317)

CAUTION! This email came from outside of Morehouse School of Medicine. Exercise extra caution clicking links and opening attachments from any and all senders.

Hi @tsimoneauxhttps://github.com/tsimoneaux

you can run STAR without annotations, but then you cannot use --quantMode GeneCounts option, which requires knowledge of gene/transcript coordinates.

Cheers Alex

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/alexdobin/STAR/issues/1317#issuecomment-900653628, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AHHEWH7CIBEEMSDQ5HDL6SDT5LKJZANCNFSM5BRZV4OQ. Triage notifications on the go with GitHub Mobile for iOShttps://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675 or Androidhttps://play.google.com/store/apps/details?id=com.github.android&utm_campaign=notification-email.

[alexdobin]

Hi @tsimoneaux

This problem is likely due to insufficient RAM. Please try to run STAR without these parameters --twopassMode Basic --sjdbGTFfile /DATA/REFERENCES/GRCH38_MainChrs/Homo_sapiens.GRCh38.104.gtf (you do not need this one at all if you used this file at the genome generation step) --outFilterType BySJout --chimOutType Junctions SeparateSAMold WithinBAM SoftClip --chimOutJunctionFormat 1 --chimSegmentMin 15 --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM GeneCounts

Then add these parameters one by one to see which ones are causing the problem.