Trimming with sliding window before alignment

[blancha]

Hi,

I'm just wondering how trimming with a sliding window, with a tool such as Trimmomatic, will affect alignment.

If Trimmomatic cuts sequences in the middle of the reads, will that not negatively affect the quality of the alignment, rather than improve it?
An aligner, such as Bowtie2, would not be aware of the missing bases in the middle of the read.

Here is an example of a trimming command from the Trimmomatic website with the sliding window parameter.

java -jar trimmomatic-0.27.jar PE s_1_1_sequence.txt.gz s_1_2_sequence.txt.gz lane1_forward_paired.fq.gz lane1_forward_unpaired.fq.gz lane1_reverse_paired.fq.gz lane1_reverse_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Scan the read with a 4-base wide sliding window, cutting when the average quality per base drops below 15

I don't understand how an aligner such as Bowtie2 can generate a better alignment with missing bases in the middle of the reads, rather than keeping the reads, knowing that they are of lesser quality, in the middle of the reads.

Of course, if the trimming reduces the length of the reads below 36, the reads are dropped, which is fine. I am talking about the cases where the entire read is not dropped.

Thank you.

[kmcarr]

Scan the read with a 4-base wide sliding window, cutting when the average quality per base drops below 15

I don't understand how an aligner such as Bowtie2 can generate a better alignment with missing bases in the middle of the reads, rather than keeping the reads, knowing that they are of lesser quality, in the middle of the reads.

Trimmomatic (or any trimming tool employing this strategy) does not remove bases and the middle while retain reads on either end of a read. What the sliding window trimming does is scan along the read in the 5'->3' direction calculating the average quality over the window size you have defined. Once it encounters a window with an average quality below the threshold you have specified it removes all bases in that window and beyond to the 3' end of the read, effectively trimming the read back to the point just before the sliding window fell below your threshold.

[blancha]

Great. Thanks for the answer. That is exactly what I wanted to know. It's not very clear from their website or their paper, but your explanation makes perfect sense.