Difference in sequence length: is it a problem?

[yanxianl (Yanxian Li)]

Hi,

@microbiotaphyto

There is a 100 nts difference between forward and reverse reads.

The read quality looks good. It's not common that the reverse reads are much shorter than the forward reads. Do you know what happened during the sequencing? Did the sequencing stop earlier than it should have been?

@microbiotaphyto

Is it a problem?

Based on your quality plot, if you trim your primers and truncate the last 10 nt of both reads you should end up with 430-440 nt after merging forward and reverse reads. If the expected amplicon size of your study + 20 nt (the recommended minimum overlapping length) is less than 430-440, you should be fine. If not, you can discard the reverse reads and use the forward reads only at the cost of lower taxonomic resolution.

@microbiotaphyto

Should I trim those extra 100 nts from the forward reads?

No. You just need to trim the last few nucleotides, say 10 nt.

@microbiotaphyto

Also, I know I should trim out my primers, but how can I check if there are primers to trim and their length?

You can ask your sequencing service provider for the primer sequences. To check if the primer sequence is present in your reads, which I suspect is, you can check this forum post:

Finding Primers In Raw Files and Quality Control

Hi @Pauline_Trinh, You will need to just check the files to be sure, though asking the sequencing center may be more straightforward (especially if your primers contain degenerate bases). If you have the raw .fastq files (before importing into QIIME2) and your primer does not contain degenerate bases, you could type the following command into your terminal (replacing ACGTACGT with your actual primer sequence): grep 'ACGTACGT' path-to-your-fastq-file.fastq | wc -l That will list the number of…