RSEM error
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2.1 years ago
glady ▴ 320

Hello,

I'm trying to use RSEM for a paired-end data to map it against human reference with STAR aligner. After, trimming the adapter sequences with cutadapt, when I try to align the reads with RSEM, with the following command;

rsem-calculate-expression -p 6 --paired-end --phred64-quals --output-genome-bam --star --star-path /home/Downloads/tools/STAR-2.5.3a/source --estimate-rspd --append-names sample1-R1_trimmed.fastq sample1-R2_trimmed.fastq ref_genome/human_ref sample1.expression

I get this error;

Warning: Read A00902:595:HC27FDRX2:2:2226:26476:24236 is ignored due to at least one of the mates' length < seed length (= 25)!

How can I solve this issue? Is this because the adapter trimming is not done correctly? Or there is some issue with the input RSEM commands I used?

Thanks!

mapping transcriptomics RSEM genome alignment • 1.6k views
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It's a warning, not an error. If you trimmed adapters you could also filter according to size (cutadapt has this option, other tools as well)

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Okay, thank you for the reply. So is it okay, if I ignore and proceed with this warning?

Because another issue is the diagnostic plot, where the unaligned reads are 98%. But if you open the genome.bam file in FASTQC, you can see most of the reads are present.

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98% unaligned is bad. The reads are there but they're not aligned so it's pretty useless.

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How can I solve the issue? Is it because of the warning from RSEM? or the adapter trimming was not done properly?

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Could be several things. Try to manually BLAST some unmapped reads to see where they fall (in non-coding regions, rRNA etc. another genome ...). Run fastqc after trimming to see if you have adapters left (although less likely)

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I don't see any adapters after trimming by cutadapt.

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The issue is the adapters and illumina indices, in the FASTQC report I can see some sequences as TruSeq Adapter index 15.

But even after trimming these sequences by cutadapt, I still find a poor mapping percentage!

What could be the problem? Should I try some different algorithm instead of cutadapt?

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