When I trim sequences for RNASeq, can I trim by sliding window on quality score or will the fact that the Illumina reads are now of different lengths affect the FPKM values (using cufflinks)? Should I just do a 3' and 5' trim of a specific length and leave all lengths equal?
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Not sure if there are any papers on this yet, but I don't think so.
I tried the window based trimmer ea-utils on my RNAseq datasets and realigned, getting extremely similar results to my untrimmed datasets.
I should mention this is on very short read data (35-40bp).
For longer reads with low quality ends this is of course more of an issue. -
This is unless the aligner takes base-call quality scores into account, in which case trimming should be unnecessary anyway.Comment
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Can you please elaborate on that? Why would the trimming be unnecessary?Comment
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The point of trimming low-quality base-calls is to remove bases which might be erroneous. A naive aligner would be deterred from a valid mapping by mismatches that are only due to sequencing errors. A quality-aware aligner, however, knows that a mismatch of a low-quality base is no reason to reject an otherwise good mapping and will report it. Many of the currently popular aligners work this way, i.e., they pay less attention to the low-quality ends of reads. By trimming, you basically take away from the aligner the chance to make use of this feature. However, trimming makes a hard-cut decision (everything below a quality threshold is removed) while a well-designed quality-aware aligner may make a more sophisticated, gradual, decision.Comment
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Thanks for the answer- do you know which aligners work in this way? (I am working with bowtie and cufflinks)
The reason we were thinking of trimming is because we are working with mixed communities (co-cultures) so we may have two bacteria in the same Illumina. We then align all the data to each of two genomes, allowing zero mismatches.
Given this, would your answer still be not to trim?
And just for my general knowledge- do you know if different length reads if we did trim would affect the final FPKM calculations?
Thanks!Comment
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