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Generating Read Length?

Hello! I am new to RNA seq. I quality trimmed my fastq sequences via fastX toolkit using a phred score of 30. I would like to figure out the read length after the quality trim (phred score < 30 was removed). Any help would be much appreciated.

-Nikelle

rna-seq phred-quality read-length
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updated by Ram Ram • written by nikelle.petrillo nikelle.petrillo

This kind of trimming is very severe and likely unnecessary. I assume you are aligning to a reference genome. You are probably throwing away lot of good data if you did lose a lot if bases after trimming.

See this thread for a paper referenced in there about trimming using quality: Which Phred value to use in trimming

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written by GenoMax GenoMax

You might want to consult www.rnaseq.wiki for a really nice description of the steps involved in processing RNAseq. IIRC, they cover read trimming in the appropriate section

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written by Chris Miller Chris Miller

2 answers

Run FastQC before and after trimming.

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updated by Ram Ram • written by Biomonika (Noolean) Biomonika (Noolean)

Thank you very much! One other question. Before trim, I had sequence length = 100. After trimming, I had a sequence length of 3-100. Can you make any sense of this? I think I am having a hard time figuring out what length it is measuring.

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updated by Ram Ram • written by nikelle.petrillo nikelle.petrillo

That means some reads were trimmed to a length of 3 eliminating 97 bases and you have a range of read lengths remaining that goes from 3 to 100. See my comment for your original question.

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written by GenoMax GenoMax

How about this:

sed -n '1~4p' filename.fastq | perl -ne 'chomp;print length($_) . "\n"' | sort -n | uniq -c >length.dist
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updated by Ram Ram • written by Chris Miller Chris Miller

Hi, Thanks Chris. If I were to input this, what would this be generating?

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written by nikelle.petrillo nikelle.petrillo

Let's break it down:

sed -n '1~4p' filename.fastq

Gives you every 4th line of the file (the sequence line)

perl -ne 'chomp;print length($_) . "\n"'

outputs the length of that line

sort -n | uniq -c

condenses it into a table of counts like this:

  3  98
123  99
 22  100
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updated by Ram Ram • written by Chris Miller Chris Miller

Thanks very much, that was helpful. What are the two different columns in the table?

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written by nikelle.petrillo nikelle.petrillo

Count and read length (see man uniq)

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updated by Ram Ram • written by Chris Miller Chris Miller

Chris, the fourth line of FASTQ is the quality score, not sequence (but it should be trimmed to the same length as the sequence string, so results should be the same).

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written by harold.smith.tarheel harold.smith.tarheel
The command gives every 4th line, starting with the 1st. Come to think of it, that should be 2~4p, right, because of the header?
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written by Chris Miller Chris Miller

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