/ 
FASTX-Abi-0.04
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/ FASTX::Abi

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 0.04

SYNOPSIS

use FASTX::Abi;
my $filepath = '/path/to/trace.ab1';

my $trace_fastq = FASTX::Abi->new({ filename => "$filepath" });

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

TEST

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.

Sanger trace AB1

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)

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.

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";

_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