I have a questions regarding how to remove primer before running data2 (or use the trim command to remove primer). The primer set I used is 515f/806r The sequencing center told me that :
"the primers will vary a bit in size, that is, by 1, 2 or 3 bases. The reason is that Illumina’s software does not do very good when the first few bases are the same, which is the case when we use the same universal primers for all the samples. So we (and others) have approached this by adding 1, 2, or 3 Ns at the beginning of the primer. That helps the software make the right base call and helps us obtaining more real data. "
So the primers are look like the following image:
So as a result, the primer F4 has three additional N compared to F1, F3 has two additional N compared to F1, and F2 has one additional N compared to F1. The final sequence length also varied from 291-294 bp. The sequencing center told me that they have already remove barcode. So what I need to remove is the primer (showing the letter after dash in the image).
I saw in the forum that people use --p-trim-left to remove primer. However, in my case, the position of my forward primer varied from 19 to 22 and the reverse primer varied from 20 to 23. If I using the following command : --p-trim-left-f-22 and --p-trim-left-r-23. They can remove all the primer but for some samples, additional Oligonucleotide can also be removed. If I did this, all the sequence after trim can also be different length since their original length is different and the length being remove is the same.
Do you think this is a good way (using Data2) to remove primer in my situations? Will this affect the other following analysis. Can you please give me some suggestions regarding how to remove the primers in my cases (either using Data 2 or other primer removal program).
Thank you for your help:smiley:.