Died at ../../../NovoGraph-1.0.0/scripts/BAM2ALIGNMENT.pl line 246.

[SAMtoBAM]

Hi there,

I have run the following minimap2 -a -x asm20 ../ref/S288C_reference_sequence_R64-2-1_20150113_renamed.fsa ../homozygous_genomes/RENAMED/AAB.FINAL.canu_RENAMED.fa | samtools view -Sb - > AAB.bam Mapping one genome to a reference I checked and all contigs map When I run the next step: perl ../../../NovoGraph-1.0.0/scripts/BAM2ALIGNMENT.pl --BAM AAB.bam --referenceFasta ../ref/S288C_reference_sequence_R64-2-1_20150113_renamed.fsa --readsFasta ../homozygous_genomes/RENAMED/AAB.FINAL.canu_RENAMED.fa --outputFile intermediate_files/AlignmentInput.txt I get Died at ../../../NovoGraph-1.0.0/scripts/BAM2ALIGNMENT.pl line 246.

Any idea what the problem is?

Thanks Samuel

[evanbiederstedt]

Hi Samuel

It's a somewhat weird error, as there should be some sort of informative messages for most die if checks.

https://github.com/NCBI-Hackathons/NovoGraph/blob/master/scripts/BAM2ALIGNMENT.pl#L236-L252

unless($alignment_reference_noGaps eq $supposed_reference_sequence)
{
    print "Reference mismatch for read $readID\n";
    #print "\t", "CIGAR: ", $inputAlignment->cigar_str, "\n";
    # print "\t", "Softclip remove: ", join("\t", $remove_softclipping_front, $remove_softclipping_back), "\n";         
    #print "\t", $alignment_reference, "\n";
    #print "\t", $alignment_read, "\n";
    print "\t", "firstPos_reference_0based: ", $firstPos_reference_0based, "\n";
    print "\t", "REF: ", substr($alignment_reference_noGaps, 0, 20), " .. ", substr($alignment_reference_noGaps, length($alignment_reference_noGaps)-10, 10), " ", length($alignment_reference_noGaps),  "\n";
    print "\t", "REF2: ", substr($reference_href->{$chromosome}, $firstPos_reference_0based, 20),  " ", length($supposed_reference_sequence), "\n";
    #print "\t", "ALG: ", $supposed_reference_sequence, "\n";
    print "\t", "strand: ", $strand, "\n";          
    print "\n";
    $warnings++;
    die if($warnings > 0);
    return undef;
}

From what I see, line 246 is commented out. Do you have any more information (or data) to help debug? You also might want to print the variable $warnings to see if that returns something. If so, that could help you fix the issue.

[SAMtoBAM]

Looks like the issue is with downloading the v1.0 tar.gz! Also perhaps why it was previously asking for Bio::DB::HTS..

Running now with the latest scripts, appears to be working

Thanks

[evanbiederstedt]

Glad this was solved.

In principle, the errors in the scripts are meant to give users some clue as to how to debug the issue, if there are issues. I didn't know how to address this error though, so I'm glad it was traced back to an incomplete download.

Thanks, Evan