pcr.seqs [ERROR]

[giovannapires]

I get the following issue whenever I try to run pcr.seqs with a custom region from the UNITE database:

[ERROR]: seqs are not aligned. When using start and end your sequences must be aligned.

I can follow the MiSeq SOP right up to this point. Then, I start getting this error. I tried aligning the ITS primer + one of my sequences with UNITE_v6_sh_97_s.fasta in R Studio using rBLAST. After figuring out the s.start e s.end, I type:

pcr.seqs(fasta=UNITEv6_sh_97_s.fasta, start=100, end=400, keepdots=F)

And get the same error again and again. What am I doing wrong?

P.S.: I'm using Ubuntu 24.04 LTS WSL on Windows 10.

[pschloss]

Hi - the unite databases are not aligned. You would have to align the unite sequence to each other using something like clustal or muscle and then use that as the reference alignment.

Pat