Oligo file construction from a study

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1

Hello,
I am trying to recreate and study where they used modified 515f and 806r primers. I am creating the oligos files but the sequences given for a sample set are for example:

515 forward = GCCTTGCCAGCCCGCTCAGGTGTGCCAGCMGCCGCGGTAA;
806r = GCCTCCCTCGCGCCATCAGCTCGCGGGGACTACVSGGGTATCTAAT;

I did a search and found which are actually the primer sequences within these two strings, but we are clearly having adaptors and linkers in both forward and reverse. Should I list both barcodes in the file (basically two barcodes for one sample)?

Thank you

2

You can remove adapters and linkers within mothur using the linkers and spacers parameters in the https://mothur.org/wiki/Oligos_File#Linkers.

If you post a sample sequence you want these primers removed from, I can give you a more detailed response?

3

Here are a couple of of reads from pyrosequencing in one sample

GN85KWK04I5YLV rank=0000045 x=3641.0 y=1249.0 length=168
GGGACGTACCGGGTAGTCTAATCCCTTTCGCTCCCCTCGGCTTTCGTCCATGAGCGTCAGTTATGGCCCAGCAGAGCGCCTTCGCCACTGGTGTTCTTCCCGATATCTACGCATTTCACCGCTACACCGGGAATTCCCTCTGCCCCTACCACACTCAAGCCTT

the primer used with barcodes listed for them are

515 forward = GCCTTGCCAGCCCGCTCAGGT GTGCCAGCMGCCGCGGTAA; 806r = GCCTCCCTCGCGCCATCAGTGTAGGG GGACTACVSGGGTATCTAAT;

I 515 forward primer is the same for the other samples and only 806r varies in some lenght (which I believe must be the barcodes)

How should a oligo file should look like?
Thankyou

4

Hi,

I don’t see either primer sequence in your sequence. Is this from a raw read or has it already been trimmed? Do you know which primer they sequenced off of? I assume it’s the 806r since you think its the one with the barcode

Pat

5

Yes, they listed as a raw reads but I as you say I think that they have already trimmed the barcodes when demultiplexing, because I got 12 different fasta files, one per each type of sample.

I can see that most of the reads start with the reverse primer 806R = GGACTACHVGGGTWTCTAAT, I ended up doing a trial were I just created a oligo file listing this sequence as forward and did not list the barcodes. And it seems to work.

Maria

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