Restriction enzyme sequence showing up after process_radtags

[Emily Dziedzic]

I'm having issues with some ddRAD sequencing results.

pstI and mspI were used on genomic DNA and there was NO size selection, unfortunately. Sequencing was performed in a single NovaSeq lane.

The first 3 bases in the reverse reads were very low quality, often 'N' so I trimmed them using trimmomatic and HEADCROP:3 before running process_radtags to avoid dealing with the restriction enzyme not being found.

I used the following command:

process_radtags -i gzfastq -1 r1_otter_ready.fq.gz -2 r2_otter_ready.fq.gz -o dir -e pstI -b barcodes.txt -t 105 --adapter-1 AGATCGGAAGAGCGGT --adapter-2 AGATCGGAAGAGCGTC -c -q -r --score-limit 20

The barcodes are of varying lengths, not sure if that matters.

I then ran fastqc on the resulting reads, and it looks like the restriction enzyme cut site sequence 'TGCAG' now appears at the beginning of every forward sequence. Shouldn't process_radtags have removed it?

Many thanks for any and all help!

Cheers,

Emily

[Catchen, Julian]

Hi Emily,

 

By default, process_radtags does not remove the restriction enzyme cutsite remnant, just checks that it is intact (and corrects it if it is nearly intact). If you want to remove it, you can add the cutsite onto the end of your barcodes so it is trimmed along with them, but I recommend keeping the cutsite.

 

Best,

julian

[Emily Dziedzic]

Ah! Okay! That makes everything fall into place. Thank you!

Is there a diagram of process_radtags order of operations and what is done at every step in detail? The manual is comprehensive, but it would be useful to know what happens at every step in more detail.

Many thanks!

Cheers,

Emily