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 reactions. I gave a presentation about a month ago about the process that Oxford Nanopore Technology use in their strand-switching cDNA kit, which may be useful for ...
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 - unless that nucleotide does not match the primer. Since - is normally used to represent gaps, I recommend using . instead for matching nucleotides.
I know you're looking for ...
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" your primer within the sliding window to assess the "beginning" and the "end" and remove "artifactual" hairpins. Its doable via a script and you would automate ...
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):
2) Target Start
3) Target Stop
4) Target Name (change which affected everything downstream in counting)
5) 5' Primer Start
6) 5' Primer Stop
7) 5' Primer ...
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. This prevents the variants from being called based that are synthetic but if you have an overlapping amplicon it will call bases from those reads.
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 local alignments of sequences to references. If the number of mismatches are not too large (as would be expected for primer sequences), a local alignment will ...
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 downloaded files with a tool like dos2unix.
On a whim, I tried adding a description to the FASTA header:
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 itself in the vast majority of reads.
I wouldn't clip the bam file, I'd clip either the fastq, or clip within bcl2fastq while making the fastqs.