gear-genomics/pearl

Patching references via trace assemblies

pearl's Issues

Chromatograms that inclued data outside of reference fasta leads to nonsensical results

I use forward and reverse sequencing on amplicons using M13 tails on degenerated primers (with inosine). This leads to my dendrograms including the degenerated primers regions. To avoid including this unreliable data in my next analysis, I use a reference fasta corresponding only to the region between the primers.

But if I upload a chromatogram that has data outside the reference, the results are none-sensical :

Removing this region resolves the problem.

I don't know if this is a bug. For me it is a issue since I must do a little more work to find and remove the primers' data in every chromatogram, and I was expecting the algorithm to understand that it must stick to the reference fasta when aligning the sequences, and not to the chromatograms. Also, it might mean that the alignment is perform only from one extremity of chromatogram. In this case, resolving the problem will mean a little more work...

Limit of number of files in the web application?

I was wondering if there is a limit to the number of files that can be aligned with Pearl. I had tried 26 files and it seems to have some kind of problem. I am getting the message "Network error"

Reference sequence can't contain degenerate/heterozygous bases

If the reference sequence contains a degenerate/heterozygous base (W, S, M, K, R, Y, etc.), it gives the error "FASTA file contains nucleotides != [ACGTN]."

As a quick and dirty fix, it could map these bases to N. The best fix would be to use the bases according to their meaning, e.g. C or T for Y.

saving sequence exports "reference only" bases

I aligned two chromatograms of the same amplicon (forward and reverse), with a reference fasta from another source to observe the covering of my sequencing and to exclude the PCR primers (the sequencing was performed using M13 tails).

When using "Save user sequence (FA)" to retrieve the sequence after treating the conflicts, I realized that the exported sequence include a part that was only determined by the reference fasta I used. Even if there is some supporting data in one of the chromatograms (see image below), it was not included in the analysis (maybe it is also a problem).

Strangely this does not happened in 3' of the sequence (see image below), were there is also supporting data but no reference-only bases in the exported sequence.

I hardly understand how the trimming is done, by the way. Quality remains rather high after the reverse chromatograms that is problematic.

As a temporary work-around, I manually deleted the undesired bases using "Treat as not sequenced", but it is a bit long...

Forward and reverse directions are sometimes assigned incorrectly

I suspect this is a bug in Tracy rather than Pearl, but Pearl is where I see it manifested. Sometimes when I have Pearl analyze a pair of ab1 trace files, it incorrectly assigns the forward sequence as reverse and the reverse sequence as forward, leading to a consensus sequence that is the reverse compliment of the true sequence. I'll attach two trace files that cause this problem. Note that these are actual high quality trace files of DNA barcodes for insects, which is a fairly standard application for a trace assembler. It's a bit mysterious that Pearl/Tracy doesn't let you manually assign the forward and reverse directions for the traces, as this is standard in all other trace assemblers I've used. I'm curious how you're automatically assigning the directions.
traces.zip

Accept fasta files with filename extensions ".fas" and ".fasta", not just ".fa"

When I tried to upload a reference sequence into Pearl, it gave me an error message. I wasn't sure what the problem was at first, but then I figured out it was because the filename extension was ".fas", not ".fa" as the program was expecting. Personally, I've used both ".fas" and ".fasta", but never ".fa".