Welcome to the forums, @Mustafa_Talib,
Yes, you can use the reverse read!
That looks like binned quality scores from the Nextseq platform. You can read more here:
You can also search for 'nextseq quality' or 'binned quality scores' to find related questions on the forums that have already been answered!
Do you have any other questions?
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The issue is that most of my sequences are falling below a value of 25. I truncated using 270 for Forward and 140 for reverse to include as many high-quality reads as possible; however, the sequence did not merge well. As I understand from the post you refer to, I have to try different lengths for truncation, but this will mean I have to violate the quality score matter. which will affect my subsequent analysis because I will include a low-quality sequence that might not be a real representation of the sequence region.
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Hello!
I hope it is ok that I will jump in. Did you check the dada2 stats?
If you are losing most of the reads at the filtering step, then indeed the issue is in the quality of the reads. However, if you lost a lot at the merging step, that means that your truncation values are too strict, most likely, for the reverse reads. So, first of all, I would increase it for the reverse reads to make sure that the sequences overlap (12 bp ba default) enough for merging. Then you can check again dada2 stats to check if you are still losing reads, and if yes, then at which step. If needed, you can play with quality score thresholds to find the compromis between desired quality and the number of reads retained after dada2.
BTW, you may want to remove the primers from the sequences before dada2.
Best,
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