NAME

FASTX::Abi - Read Sanger trace file (chromatograms) in FASTQ format. For traces called with hetero option, the ambiguities will be split into two sequences to allow usage from NGS tools that usually do not understand IUPAC ambiguities.

VERSION

version 1.0.1

SYNOPSIS

use FASTX::Abi;
my $trace_fastq = FASTX::Abi->new({ filename => '/path/to/trace.ab1' });

# Print chromatogram as FASTQ (will print two sequences if there are ambiguities)
print $trace_fastq->get_fastq();

BUILD STATUS

The source from GitHub is tested using Travis-CI for continuous integration. Check also the CPAN grid test for a better estimate of build success using CPAN version of interest.

INSTALLATION

Via cpanminus:

# Install cpanminus if you don't have it:
curl -L https://cpanmin.us | perl - --sudo App::cpanminus

# Install FASTX::Abi
cpanm FASTX::Abi

Via Miniconda https://docs.conda.io/en/latest/miniconda.html:

conda install -y -c bioconda perl-fastx-abi

HETERO CALLING (IUPAC AMBIGUITIES)

When Sanger-sequencing a mix of molecules (i.e. PCR product from heterozigous genome) containing a single-base polimorphisms, if the .ab1 file is called using the hetero modality the sequence stored in the file will contain ambiguous bases (i.e. using DNA IUPAC characters).

This module is designed to produce NGS-compatible FASTQ, so when ambiguous bases are detected the two "alleles" will be split into two sequences (of course, if more SNPs are present in the same trace, the output cannot be phased). The image below shows a trace file (.ab1) with a valid variant and a low quality end.

METHODS

new()

When creating a new object the only required argument is filename.

# Trimming is based on Bio::Trace::ABIF->clear_range()
my $trace_fastq = FASTX::Abi->new({
  filename   => "$filepath",
  min_qual   => 22,
  wnd        => 16,
  bad_bases  => 2,
  keep_abi   => 1,      # keep Bio::Trace::ABIF object in $self->{chromas} after use
});

# Raw sequence and quality:
print "Raw seq/qual: ", $trace_fastq->{raw_sequence}, ", ", $trace_fastq->{raw_quality}, "\n";
# Trimmed sequence and quality:
print "Seq/qual: ", $trace_fastq->{sequence}, ", ", $trace_fastq->{quality}, "\n";

# If there are ambiguities (hetero bases, IUPAC):
if ($trace_fastq->{diff} > 0 ) {
  print "Differences: ", join(',', @{ $trace_fastq->{diffs} }), "\n";
  print "Seq 'A': ", $trace_fastq->{seq1}, "\n";
  print "Seq 'B': ", $trace_fastq->{seq2}, "\n";
}

Input parameters:

filename, path

Name of the trace file (AB1 format)

trim_ends, bool

Trim low quality ends (true by default, highly recommended)

min_qual, int

Minimum quality value for trimming

wnd, int

Window size for end trimming

bad_bases, int

Maximum number of bad bases per window

get_fastq($sequence_name, $fixed_quality)

Return a string with the FASTQ formatted sequence (if no ambiguities) or two sequences (if at least one ambiguity is found).

If no $sequence_name is provided, the header will be made from the AB1 filename. If $sequence_name is defined and contains spaces, they will converted to underscores.

The $fixed_quality is a user provided fixed quality value for each base printed. Can be an integer (10 < x < 40), or a single char. In the first case it will be encoded as quality score (values above 93 will all be rendered as ~), in the second case the character will be used as quality score. If not supplied the original quality of the chromatogram will be used (that will be very low in SNPs positions).

# Use 40 as quality for each base of the trace:
$trace->get_fastq(undef, 40);

get_trace_info()

Returns an object with trace information:

my $info = FASTX::Abi->get_trace_info();

print "Instrument:            ", $info->{instrument}, "\n";
print "Version:               ", $info->{version}, "\n";
print "Average peak distance: ", $info->{avg_peak_spacing}, "\n";

rc()

$trace->rc();

Reverse complement the chromatogram sequence.

merge()

Merge two chromatograms if overlapping returning the merged sequence.

my $merged = $trace->merge($other_trace);

_get_sequence()

Internal routine (called by new()) to populate sequence and quality. See new()

SEE ALSO

This module is a wrapper around Bio::Trace::ABIF by Nicola Vitacolonna.

AUTHOR

Andrea Telatin <andrea@telatin.com>

COPYRIGHT AND LICENSE

This software is Copyright (c) 2019 by Andrea Telatin.

This is free software, licensed under:

The MIT (X11) License

cpanm FASTX::Abi

perl -MCPAN -e shell
install FASTX::Abi