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4 votes
Accepted

What is a poly(T) primer anchor sequence?

As shown in figure 1 of that paper, the anchor sequence is additional sequence that is added to the end of the polyT primer that provides a known and non-target binding site for additional PCR ...
  • 12k
3 votes
Accepted

Aligning sequence and comparing it against primer

If I understand you correctly, after your most recent edit, you have 5,000 sequences that are 63 bp long and in each you simply want to replace the nucleotides from 21 through 43 with ...
2 votes
Accepted

programmatic secondary structure prediction for >36-mer DNA oligonucleotides

If you really insisted on using Primer3 you could perform a 36bp sliding window analysis. You would submit your primers in 36bp 'chunks' with a step size of say 3 bp. You would need to "circularise" ...
  • 8,005
2 votes
Accepted

What is the "primer masterfile" format?

this is the swift standard primer file. You are a bit off in your interpretation of the columns (seems like you missed one as there are 12 columns, not 11 which threw everything off): 1) Chromosome ...
  • 601
2 votes

Hard-clip primers in a bam file

Another option would be to adjust the quality of the primer bases to be below the threshold that your variant caller uses. In my case I have "masked" the primer bases by dropping the quality to 0. ...
  • 2,576
1 vote

Do you know a program/script to count how many sequences have mismatches on each position of my primers?

I'm amazed at your tenacity in getting Excel to do what you want; that looks like many hours of work to count mismatches. The hammer I use at the moment for this sort of stuff is LAST, which will do ...
  • 12k
1 vote

Primer-Blast Custom Database [error]

I just copied your FASTA sequence into a file (on Mac OS) and uploaded it to Primer BLAST, the search did not give an error. If the error is caused by the line endings, maybe you can fix your ...
  • 353
1 vote

Hard-clip primers in a bam file

I know that the primer sequences are part of the genome and a variant there is no less valid than a variant anywhere else, You won't see variants under PCR primers. You get the primer sequence ...
  • 1,722
1 vote
Accepted

Hits in Primer-BLAST not found with programmatic blastn query

I've found the problem, and it's way less interesting than I initially thought. Essentially, my "eValue" was way too low. Instead of -evalue 100, I'm now using <...
  • 192

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