STAR alignment , remove duplicate, RSEM

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jacs

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May 27, 2022, 6:34:52 AMMay 27
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Hi Alex,

I am trying to use RSEM with BAM files from STAR alignment but I getting error when using BAM files (duplicate removed BAMs) (at Step 3).

I tried following command:
step 1.
 STAR --runMode inputAlignmentsFromBAM --bamRemoveDuplicatesType  UniqueIdentical  --inputBAMfile Aligned.soretedByCoordinate.bam
step 2.
 samtools view -b -F0x400 Processed.out.bam > Processed.out.noDupl.bam
step 3.
rsem-calculate-expression --bam  --no-bam-output -p 8  --paired-end --forward-prob 1 Processed.out.noDup.bam  /database/RSEM/rsemrefgenome  xy.rsemQuant

rsem-parse-alignments /database/RSEM/rsemrefgenome xy.rsemQuant.temp/xy.rsemQuant xy.rsemQuant.stat/xy.rsemQuant Processed.out.noDup 1 -tag XM
[E::hts_open_format] fail to open file 'Processed.out.noDup'
Cannot open Processed.out.noDup! It may not exist.
"rsem-parse-alignments /database/RSEM/rsemrefgenome xy.rsemQuant.temp/xy.rsemQuant xy.rsemQuant.stat/xy.rsemQuant Processed.out.noDup 1 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!


Is there any other way to use STAR with BAM files (duplicated removed BAMs) for read counts?

Thanks!

With regards,
jacs

Alexander Dobin

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Jun 6, 2022, 12:54:23 PMJun 6
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Hi jacs,

it should be possible, I think RSEM just does not see the BAM file for some reason.

Cheers
Alex

jacs

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Jun 8, 2022, 8:53:22 AMJun 8
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Thank you very much Alex!

How to use duplicated removed BAM file (Processed.out.noDupl.bam) in STAR for read counts? It would be great help if you can send me STAR command.

Regards,
jacs

Alexander Dobin

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Jun 10, 2022, 10:10:43 AMJun 10
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Hi Jacs,

what you are doing on STAR side is correct.
This is RSEM error:
fail to open file 'Processed.out.noDup'
Cannot open Processed.out.noDup! It may not exist.


It looks like RSEM cannot see the file.

Cheers
Alex

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