Dear all,

first of all, thanks a lot for this nice software. I was searching for a good and free solution for editing and assembling Sanger reads since long and might finally be there.

I have the following common situation: I have forward and reverse Sanger chromatogram reads (ab1) for the same sequence. What I want to do is to align forward and reverse sequence and check for incorrect base calls etc., looking at both chromatograms at once. After finishing the editing, I want to merge the reads to end up with one sequence for the sample. I need to do this in an efficient way for many samples.

My best attempt so far is to map all ab1 chromatograms to a reference sequence, which nicely determines forward and reverse sequences and shows all chromatograms at once. Perfect so far. From now on, I gloriously failed to produce one fasta-file that contains one merged sequence per sample.

(1) I managed to export a consensus sequence for the whole reference mapping alignment which is not what I need. (Export consensus)

(2) I produced a fasta file that contains all sequence, i.e. all forward and reverse sequences. (Export alignment without chromatogram)

(3) I invested some hours to try existing workflows and to create my own using "read sequence", "mark sequence by name", "group", and "write fasta/alignment" etc., however I can't figure out how to do it.

My ab1-files follow the following format: <ID>_<primer>.ab1 (e.g. 1234_ar_f.ab1). In Geneious, there is an option like "assemble sequences by 1st part of the name separated by '_' (underscore)", or similar.

Is there any way to solve this isse or are there even workflows around that can do such a thing from regular expressions on the file name or something?

I want to use UGENE in a course with students, soon. Any help is greatly appreciated!

Best,
Jonas

Dear Dmitrii,

thanks a lot for the fast response! I'll try to clarify:

I need a multi-fasta file (multiple sequences in one file) with consensus sequences of forward and reverse reads of each sample.

I have reads of the same gene and from the same sample in forward and reverse direction for many samples. The sample can be identified by an ID in the sequence name (<ID>_<primer>.ab1). Typically, the ends of the amplified region are only represented in good quality by either the forward or the reverse read. That results in two nearly identical sequences for each sample, only that the beginning and the end is represented by only one sequence. I attached a screenshot of this situation (see the overview at the bottom). In the example, the sample IDs would be 02, 04, and 05. The samples come from different individuals/species. In the resulting fasta file, there should consequetly be 3 sequenes (>02, >04, and >05) covering the whole range of forward+reverse read. Ideally, ambiguities would be translated into IUPAC codes (N, R, Y, S, ...).

Thanks for your help again!

Best,
Jonas

Sorry for the confusion.  I guess a small worked example would be best to explain:

After mapping my reads to a reference, I can export a fasta file that contains all reads as separate sequences (excluding the reference):


exported_fasta.fa
=~=~=~=~=~=~=~=~=~=~=~=~=~=
>individual1_gene1_Primer1_fwd
AAGGCTCTGGCTAGCTTGAAACC-----
>individual1_gene1_Primer2_rev
-----TCTGGCTAGCTTGAAACCGCTSG
>individual2_gene1_Primer1_fwd
AAGGATCTGGCTAGCTTGAAACC-----
>individual2_gene1_Primer2_rev
-----CCTGGCTAGCTTGAAACCGCTSG
=~=~=~=~=~=~=~=~=~=~=~=~=~=


What I need is a fasta file that contains the iteratively build consensus sequences of forward and reverse read per individual/sample, which would be obtained by the following steps:
1. filter fasta for a single individual
2. build consensus sequence of forward and reverse read(s)
3. repeat for all individuals
4. write fasta that only contains the consensus sequences.

The result would look like this:

final_fasta.fa
=~=~=~=~=~=~=~=~=~=~=~=~=~=
>individual1_gene1
AAGGCTCTGGCTAGCTTGAAACCGCTSG
>individual2_gene1
AAGGAYCTGGCTAGCTTGAAACCGCTSG
=~=~=~=~=~=~=~=~=~=~=~=~=~=


Note that site number 6 in the sequence of individual2 has the IUPAC degeneracy 'Y'.

It would be even better, if the final fasta could be directly saved without the intermediate step of the reference-mapping-fasta.

I hope that makes things clearer. This should be a very general issue for everybody who sequences forward and reverse reads for many specimens and a desired function.

Meanwhile, I found an R-package that can do the trick: sangeranalyseR
They have a manual page for the issue on sangeranalyser[dot]readthedocs[dot]io (Advanced User Guide - SangerAlignment)

However, since I plan to use UGENE with students in a course, I try to avoid using too many different software packages, in particular for presumably easy tasks like this one.

Best,
Jonas

Ok, I see. In that case, with the students, I will process the reads sample by sample. For productive work, I'll probably map all reads against a reference and use custom scripts to handle the resulting fasta that I "export without chromatograms" and look forward to version 37.

Thank you very much for your help!

Hello,
I'm not sure now, you can check the state of this issue here:
https://ugene.dev/tracker/browse/UGENE-6881