Question: illumina PE quality control - NGS: QC & Manipulation
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gravatar for sumudu_rangika
2.2 years ago by
sumudu_rangika • 0 wrote:

Hi,

I have a PE illumina miseq data set (separate forward-R1 & reverse-R2) of a WGS of a parasite. I'm using a local instance of galaxy. I have run FASTQC on sequencer given data sets and decided to do some quality control since quality drops towards the end of reads.

I have few doubts to clear and appreciate if galaxy team or anyone in the forum could help me to solve them.

1) According to FASTQC I decided to quality trim reads using FASTQ quality trimmer (sliding window=5, step=2, avg quality=20). Since quality degrades towards 3' end should I trim 3' end only. Is this Ok?

2) I have seen suggestions to perform quality control for PE data sets (R1 & R2) together & not separately. Isn't it correct to perform QC on R1 & R2 separately?

3) If so, I have to use another tool instead of FASTQ quality trimmer. Would it be a good option to use trimmomatic or Trim galore?

4) Later I used Filter FASTQ to filter reads <70bp; If I use R1 dataset only should I use yes/no to "This is paired end data" option? Can I select multiple data sets option in filter FASTQ to choose both R1&R2 together?

Thanks in advance, Sumudu

ADD COMMENT • link • modified 2.2 years ago by Jennifer Hillman Jackson ♦ 25k • written 2.2 years ago by sumudu_rangika • 0
1
gravatar for Devon Ryan
2.2 years ago by
Devon Ryan • 1.9k
Germany
Devon Ryan • 1.9k wrote:
  1. A quality of 20 is overkill for many use cases (unless you're trying to do assembly maybe, I can't give advice there).
  2. Only ever use trimmomatic, or trim galore, or a similar program with paired-end datasets. I would also suggest simply removing fastq quality trimmer, since inevitably having it there will lead someone to try to use it with paired-end data and get completely weird results.
  3. See above
  4. Both trimmomatic and trim galore have options to filter out reads below a given size, just use them. Regarding the "Filter Fastq" tool, it looks like the "paired end" option somehow signals that the paired-ends have been merged together, which wouldn't be the case for you.
ADD COMMENT • link written 2.2 years ago by Devon Ryan • 1.9k

Thank you very much Devon.

ADD REPLY • link written 2.2 years ago by sumudu_rangika • 0
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