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SRA sequence, FastQC, RNA seeq

Hello,

I have downloaded SRA data from NCBI and converted it into Fastq file (Pair end sequences), then I analysed the sequences using fastqc. The following results I got, which I think ok. But still confuse. Can anybody shade some light on this aspect. (Per base sequence quality http://dropcanvas.com/#aG53s1AtEBgLv1)

##FastQC    0.11.2
>>Basic Statistics  pass
#Measure    Value
Filename    PPlf.fastq
File type   Conventional base calls
Encoding    Sanger / Illumina 1.9
Total Sequences 24000000
Sequences flagged as poor quality   0
Sequence length 75
%GC 44

Can I use fastq sequences derived from SRA format directly for assembly and scaffolding purpose? Or else I will have to do pre-processing like removal of low quality reads,trimming of low quality bases,adapter removal?

regards

rahul

rna-seq fastqc assembly sra
• 2,455 views • link
updated by Ram Ram • written by Rahul • Rahul

1 answer

It's usual to see a base sequence quality drop at the end of long reads, it means that there are probably some sequencing errors there. In your case, I would use trimmomatic* to remove low quality reads before doing anything.

*with this option only (no need for extensive trimming if everything else is fine in fastqc):

SLIDINGWINDOW:4:15
• 0 views • link
updated by Ram Ram • written by Carlo Yague Carlo Yague

Thank you very much for showing interest in my post.

What about masking instead of teaming? can then I use trimmed sequences for the assembling and scaffolding?

regards
rahul

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updated by Ram Ram • written by Rahul • Rahul

Yes, you should use trimmed for assembly. Having adapters and low quality bases in your sequences will significantly impact the assembly.

Why do you want to mask instead of trim?

• 0 views • link
written by igor igor

Before 2 days back I have attended online NCBI NGS online workshop where I heard about masking over trimming.I have not gone through this paper but presenter acknowledged this paper.

Paper link: http://www.ncbi.nlm.nih.gov/pubmed/25494997

regards
rahul

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updated by Ram Ram • written by Rahul • Rahul

I have never done it and I don't think it would change much but you can try masking if you prefer. Thx for the link btw !

• 0 views • link
written by Carlo Yague Carlo Yague

That paper refers specifically to SNP calling.

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written by igor igor

yes you are right.... that was my mistake

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updated by Ram Ram • written by Rahul • Rahul

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