Amina Khan¹, Eric J Belfield², Nicholas P Harberd² & Aziz Mithani^1,*
1Department of Biology, Syed Babar Ali School of Science and Engineering, Lahore University of Management Sciences, D.H.A, Lahore 54792, Pakistan
² Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK

HANDS stands for "HSP base Assignment using NGS data through Diploid Similarity" and is a tool to characterize Homeolog-Specific Polymorphisms (HSPs) in allopolyploid genomes. HSPs are the positions in a polyploid genome where the homeologous subgenomes have different bases. HANDS2 involves comparative alignments of next-generation sequencing (NGS) reads from polyploid and diploid-progenitors onto a suitable reference sequence and uses diploid similarity to assign base-identities to subgenomes at HSP positions. HANDS2 characterizes homoeallelic base-identities with high accuracy even in the absence of RNA-seq data for one of the diploid-progenitors and supports up to ten diploid-progenitors. For queries regarding HANDS2 tool, please contact Aziz Mithani at aziz.mithani@lums.edu.pk

Download

HANDS2 can be downloaded from here.
C++ code to filter SAM and VCF files can be downloaded from here.

Current Version: v1.1.1 [16-June-2017]

Includes bug fixes, memory improvements and adds support for BAM files.

HANDS2 Inputs

HANDS2 takes the following files as input for the assignment of homoeallelic base-identities.

1. SAM/BAM file of the polyploid. SAM/BAM file can be obtained using standard alignment software such as BWA, BOWTIE or BOWTIE2. HANDS2 requires the alignment file to be position sorted (see SAMtools sort command)
2. A General Feature Format (GFF) file containing start and end coordinates for each gene/contig. This file is automatically generated by HANDS2 when a transcriptomic reference is constructed from a set of unigenes/contigs using ‘seq2ref’ command. HANDS2 only uses entries of the ‘gene’ type from the GFF file.
3. VCF files containing lists of HSPs and single base substitutions (SBSs) present in the polyploid and the diploid-progenitors respectively. These files can be obtained using standard variant calling tools such as SAMtools, GATK or FreeBayes. HANDS2 uses VCF version 4.0 or greater.
4. Base coverage files containing number of reads supporting a particular base at each position in a tab-delimited format for HSP/SBS validation (optional). These files can be generated from a SAM file using ‘coverage’ command available in HANDS2.
5. An optional list of positions in the reference (a tab delimited file containing the sequence/chromosome names, positions and reference base) to be checked for HSPs in addition to the positions present in the HSP list during pre-processing step.

Usage:

Commands and Options:

help	Display this help
assign	Assign homoeallelic base identities
coverage	Calculate the number of reads supporting a particular base at each position
seq2ref	Create an in silico reference using a set of unigenes, contigs or other sequences

assign - Assign homoeallelic base identities

This commands assigns the homoeallelic base-identities to the allopolyploid subgenomes. It uses the SAM/BAM file of the polyploid genome along with the VCF files for the polyploid and the diploid-progenitors to assign base-identity to the polyploid subgenomes. HANDS2 supports up to ten diploid-progenitors and can assign base-identities with high accuracy even in the absence of a diploid-progenitor. Base assignment can be preceded by an optional pre-processing step, which validates the lists of HSPs and SBSs provided as input. The pre-processing step can be turned on by providing the base coverage files for the polyploid and/or diploid-progenitors. An additional list of positions to be checked for HSP can also be provided in the pre-processing step.

Input Parameters

-h or -help	Display this help
-i <File>	Polyploid SAM/BAM file
-g <File>	GFF file containing gene start/end coordinates
-hsp <File>	Polyploid HSP file in VCF Format.
-snp<n> <File>	Diploid # n SNP file in VCF Format.
-bc <File>	Polyploid Base coverage file (optional). See coverage command.
-bc<n> <File>	Diploid # n Base coverage file, e.g. bc1 (optional). See coverage command.
-out<n> <File>	Sub-Genome # n output file, e.g. out1
-vcf <Boolean>	Generate VCF output (Default: TRUE). When FALSE, tab-delimited output is generated.
-sp <float>	SNP pair proportion threshold (Default: 0.05)
-pm <float>	Base pattern matching threshold (Default: 0.5)
-pa <char>	Base pattern assignment mode (M: Keep maximum proportion for a base or A: Add all proportions; Default: M)
-r <Boolean>	Rectify Assignment using reference genome (Default: FALSE)
-m <Boolean>	Merge Base Patterns before assignment (Default: FALSE)
-d <int>	Use genome <int> as distant genome (Default: <null>). Cannot be used with missing genome.

Note: At most one diploid SNP file can be missing. Use "" for the missing file.

coverage - Calculate base coverage for each position from a SAM/BAM file

This command creates a base coverage file for the given SAM/BAM file containing number of reads supporting a particular base at each position. Base with phred quality less than the specified threshold (-q) are ignored when calculating the base coverage. Base quality check can be turned off by providing ‘-q 0’ as parameter. The output is in the tab-delimited format containing the number of reads supporting bases A, T, G, C and N. This file can be used to validate the variant list during the pre-processing step of homoeallelic base assignments by HANDS2.

seq2ref - Create an in silico reference from given sequences/contigs

This command creates an in silico reference from a set of sequences which can be used as a reference sequence for the alignment of sequencing reads using standard alignment tools like BWA, BOWTIE or BOWTIE2. This command reads a multi-fasta file (containing multiple sequences in fasta format) and generates a fasta file by concatenating the sequences such that two consecutive sequences are separated by a stretch of Ns. It also generates a GFF file for the created reference.

Input Parameters

-h or -help	Display this help
-i <File>	Input sequence file (multi-fasta format)
-o <File>	Output file
-n <str>	Header for the in silico reference
-g <int>	Gap size between two sequences (Default: 200)

Examples:

Assign homoeallelic base-identities

To run the wheat example data, go to the directory where you have extracted the files and run the following command:
To run the brassica example data, go to the directory where you have extracted the files and run the following command: At most one diploid-progenitor can be designated as missing genome by not providing the SNP file (diploid # 2 in this case): Pre-processing (validation of HSPs and SBSs) can be turned off by not providing the base coverage files: To check additional positions for HSP in addition to the provided HSP file during pre-processing, provide a position file:

Calculate base coverage

To calculate base coverage file for a given SAM/BAM file, use the coverage command available in HANDS2: To turn off base quality check while calculating base coverage, use -q parameter:

Create in silico reference

To generate in silico reference from a set of contigs/cDNA sequences, use the seq2ref command available in HANDS2: