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AmBiVErT - AMplicon BInning Variant caller with ERror Truncation. For calling variants in amplicon based sequencing experiments

Project Description
Experimental Alpha Release of AmBiVErT

This program is designed for the processing of amplicon based resequencing data
generated on second generation sequencing platforms (eg Illumina HiSeq/MiSeq).

The approach used is to batch reads derived from each amplicon together in a
clustering step prior to aligning the sequence to a reference genome.

1) Cluster similar reads
2) Align for overlap
3) Align to reference target sequences
4) Consolidate calls
5) Output VCF format calls

Created by Matthew Wakefield and Graham Taylor.
Copyright (c) 2013 Matthew Wakefield and The University of Melbourne. All rights reserved.

$ ambivert --help
usage: ambivert [-h] [-f FORWARD] [-r REVERSE] [-m MANIFEST] [--fasta FASTA]
[--output OUTPUT] [--countfile COUNTFILE]
[--threshold THRESHOLD] [--min_cover MIN_COVER]
[--min_reads MIN_READS] [--min_freq MIN_FREQ]
[--overlap OVERLAP] [--hashtable HASHTABLE]
[--savehashtable SAVEHASHTABLE] [--alignments ALIGNMENTS]
[--prefix PREFIX]

AmBiVErT: A program for binned analysis of amplicon data AmBiVErT clusters
identical amplicon sequences and thresholds based on read frequency to remove
technical errors. Due to sequencing errors occuring with a more random
distribution than low frequency variants this approach reduces the number of
amplicon products that must be assigned to target regions & assessed for
variant calls. AmBiVErT overlaps forward and reverse reads from the same
amplicon and preserves local phasing information. Typical running time for
first use is several hours, which reduces to less than 10 minutes when the
hash table calculated on a previous run is supplied for analysis of subsequent
samples with the same amplicons.

optional arguments:
-h, --help show this help message and exit
-f FORWARD, --forward FORWARD
a fastq format file of forward direction amplicon
reads. May be compressed with gzip with the
appropriate suffix (.gz)
-r REVERSE, --reverse REVERSE
a fastq format file of reverse direction amplicon
reads. May be compressed with gzip with the
appropriate suffix (.gz)
-m MANIFEST, --manifest MANIFEST
an Illumina TrueSeq Amplicon manifest file.
--fasta FASTA an fasta amplicon manifest file. Sequences should be
limited to the regions to be called and exclude
primers & adaptors. This file can be provided in
addition to an Illumina manifest to specify additional
off target regions.
--output OUTPUT output of alignments with variants. Default: stdout
--countfile COUNTFILE
output of occurance counts per amplicon. Includes all
counts that map to the reference amplicon. This count
does not include reads that occured at frequencies
below --threshold <default=20>
--threshold THRESHOLD
the minimum occurance threshold. Unique amplicon
sequence variants that occur fewer than threshold
times are ignored. Default 20
--min_cover MIN_COVER
the minimum coverage at a site required to call a
variant. This parameter only has effect if it is >
threshold. Default 0
--min_reads MIN_READS
the minimum number of variant containing reads
required to call a variant. This parameter only has
effect if it is > threshold. Default 0
--min_freq MIN_FREQ the minimum proportion of mutated reads. Default 0.1
(ten percent)
--overlap OVERLAP The minimum overlap required between forward and
reverse sequences to merge. Default 20bp
--hashtable HASHTABLE
Filename for a precomputed hash table of exact matches
of amplicons to references. Generate with
--savehashtable SAVEHASHTABLE
Output a precomputed hash table that matches amplicons
exactly to references. Used to speed up matching with
--alignments ALIGNMENTS
Print a formatted text version of variant containing
alignments to a file. "-" will print to stderr
--prefix PREFIX Shorthand specification of --forward, --reverse,
--countfile and --outfile. --forward =
<prefix>_R1.fastq.gz --reverse = <prefix>_R2.fastq.gz
--outfile = <prefix>.vcf --countfile = <prefix>.counts

Additional tools for simulating amplicon data


usage: simulate_variants [-h] [--manifest MANIFEST] [--fasta FASTA]
[--read1 READ1] [--read2 READ2] [--skip_softmasked]
[--deletions DELETIONS] [--check_sam CHECK_SAM]

A script for simulating paired end reads with variants from an amplicon target

optional arguments:
-h, --help show this help message and exit
--manifest MANIFEST an Illumina TrueSeq Amplicon manifest file. Default:
--fasta FASTA a fasta file of amplicons all in the plus strand
orientation with description lines ">name chromosome
start end" Default: None
--read1 READ1 a fastq output file of forward reads. Default: stdout
--read2 READ2 a fastq output file of reverse reads. Default: stdout
--skip_softmasked dont generate variants in softmasked sequence.
Default: True
Exclude softmasked sequence when calculating start
site of read, cigar and variant detection strings.
Default: True
--deletions DELETIONS
The size deletions to insert instead of point
variants. Default: None
--check_sam CHECK_SAM
a sam file for parsing to identify entries where name
does not match the sam file mapping location and
variant strings


usage: truseq_manifest [-h] [--manifest MANIFEST] [--output OUTPUT] [--probes]
[--adaptors] [--with_probes] [--softmask_probes]

A script for converting Illumina TruSeq Amplicon manifest files to fasta files
Produces either a fasta file of target sequences without primers or a file of
primer sequences suitable for use by a trimming program (eg Nesoni clip)

optional arguments:
-h, --help show this help message and exit
--manifest MANIFEST an Illumina TruSeq Amplicon manifest file. Default:
--output OUTPUT a multi fasta output file of sequence targets. Default:
--probes output only the ULSO and DLSO primer sequences
--adaptors append Illumina adaptor sequences to the primer
--with_probes append the ULSO and DLSO sequences to the fasta target
--softmask_probes append the ULSO and DLSO sequences to the fasta target
--all_plus reorient target sequences so they are all presented on
the plus strand
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ambivert-0.5.1-cp35-cp35m-macosx_10_11_x86_64.whl (315.9 kB) Copy SHA256 Checksum SHA256 3.5 Wheel Aug 13, 2016
ambivert-0.5.1-cp36-cp36m-macosx_10_12_x86_64.whl (315.8 kB) Copy SHA256 Checksum SHA256 cp36 Wheel Jan 5, 2017
ambivert-0.5.1.tar.gz (285.5 kB) Copy SHA256 Checksum SHA256 Source Aug 13, 2016

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