SRA sequence, FastQC, RNA seeq

Question

[Rahul]

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

Answer 1

[Carlo Yague]

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

[Rahul]

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

[igor]

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?

[Rahul]

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

[Carlo Yague]

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 !

[igor]

That paper refers specifically to SNP calling.

[Rahul]

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