Gap2Seq is a program for filling gaps in scaffolds produced by genome assembly tools using short read data such as reads produced by Illumina sequencing.
Since version 3.0, it can also genotype insertions from variant calls produced by variant calling tools.
R. Walve, L. Salmela, V. Mäkinen: Variant genotyping with gap filling. In PLoS ONE 12(9): e0184608.
Gap2Seq has been tested on systems running Linux on a X86_64 architecture. Gap2Seq uses GATB library for the de Bruijn graph implementation and htslib for reading alignments for read filtering. The libraries are included in the Gap2Seq package.
Compiling Gap2Seq requires a C++11 compatible compiler (GCC 4.7+ or Clang 3.5+) and CMake version 3.1 or newer.
git clone --recursive https://github.com/rikuu/Gap2Seq
cd Gap2Seq
cd thirdparty/htslib; make; cd ../..
mkdir build; cd build; cmake ..; makeThe main script Gap2Seq and the binaries Gap2Seq-core, GapMerger, GapCutter and ReadFilter can then be found in the build directory.
Gap2Seq [parameters]
Required parameters:
-f, --filled <FASTA file> output file for filled scaffolds
-r, --reads <FASTA/Q files> short reads, several files can be specified as a list separated by ','
or
-l, --libraries <lib conf> list of aligned read libraries
-s, --scaffolds <FASTA/Q file> scaffolds with gaps
or
-g, --gaps <FASTA/Q file> pre-cut gaps
-b, --bed <BED file>
or
-v, --vcf <VCF file> variants in the reads against reference
-R, --reference <FASTA file>
Optional parameters:
-t, --threads <int> number of threads to use [default 1]
-k <int> k-mer length for DBG [default 31]
--max-mem <float> maximum memory usage of DP table computation in gigabytes (excluding DBG) [default 20]
--fuz <int> number of nucleotides to ignore on gap fringes [default 10]
--dist-error <int> maximum error in gap estimates [default 500]
--solid <int> threshold for solid k-mers for building the DBG [default 2]
--randseed <int> random seed (0 to use current time) [default 0]
--all-upper fill all bases in upper case.
--unique fill only gaps with a unique path of best length
--best-only consider only paths that have optimal length
-h, --help show this help message and exit
This example shows how to run Gap2Seq on the GAGE S. aureus data.
Download the GAGE data sets from
http://gage.cbcb.umd.edu/data/Staphylococcus_aureus/Data.original.tgz http://gage.cbcb.umd.edu/data/Staphylococcus_aureus/Assembly.tgz
Unpack the data files.
Run Gap2Seq (here we run it for the SGA scaffolds)
Gap2Seq --scaffolds Assembly/SGA/genome.scf.fasta --filled Assembly/SGA/genome.scf.fill.fasta --reads Data/original/frag_1.fastq,Data/original/frag_2.fastq,Data/original/shortjump_1.fastq,Data/original/shortjump_2.fastqThe filled scaffolds are then in the file Assembly/SGA/genome.scf.fill.fasta.
Align, sort, and index the read libraries to the scaffolds with e.g. BWA MEM.
bwa index Assembly/SGA/genome.scf.fasta
bwa mem Assembly/SGA/genome.scf.fasta Data/original/frag_1.fastq Data/original/frag_2.fastq | samtools sort -O bam - > Data/original/frag.bam
samtools index Data/original/frag.bam
bwa mem Assembly/SGA/genome.scf.fasta Data/original/shortjump_1.fastq Data/original/shortjump_2.fastq | samtools sort -O bam - > Data/original/shortjump.bam
samtools index Data/original/shortjump.bamCreate a read library configuration file, a tab-separated list with a single read library per line: bam, mean insert size, std dev, threshold
echo -e "Data/original/frag.bam\t180\t18\t40" > libraries.txt
echo -e "Data/original/shortjump.bam\t3500\t350\t40" >> libraries.txtRun Gap2Seq.
Gap2Seq --scaffolds Assembly/SGA/genome.scf.fasta --filled Assembly/SGA/genome.scf.fill.fasta --libraries libraries.txtFirst, using any insertion/variant calling pipeline, construct a VCF file of the variants in the reads against a reference genome. Then run Gap2Seq supplying it with the VCF, reference, and the reads.
Gap2Seq --vcf Assembly/SGA/variants.vcf --reference Assembly/SGA/genome.scf.fasta --filled Assembly/SGA/genome.scf.fill.fasta --reads Data/original/frag_1.fastq,Data/original/frag_2.fastq,Data/original/shortjump_1.fastq,Data/original/shortjump_2.fastqInsertion genotyping can also be combined with read filtering.
When classifying filled bases into safe and unsafe bases, all paths within the allowed interval are now considered. In the previous version, only paths with optimal length were considered. The old behaviour can still be invoked using the parameter -best-only.
ReadFilter now infers the read length. This breaks compatibility with old read library configurations.
Read filtering is slightly improved for gap filling. Unchanged for insertion genotyping.
Unmapped reads are now filtered from alignments only once per read library. Speeds up gap filling around 30% for large scaffolds.
Using the wrapper script without alignments now correctly delegates all processing to Gap2Seq-core.
Updated to GATB-core 1.4.1 and HTSlib to 1.8.
Gap2Seq.sh is replaced with a Python script, which accepts gaps/scaffolds in FASTA/FASTQ and VCF formats and reads in FASTA/FASTQ and SAM/BAM formats.
Optional per-gap read filtering when run with new script.
Gap2Seq binary is renamed Gap2Seq-core and can still be used instead of the new script.
Flanks of length between k and k+fuz are now used rather than the hard limit of k+fuz.
Switched to GATB-core 1.2.2.
Parallelization is now on gap level when run with the Gap2Seq.sh script.
Safe bases inserted into gaps are outputted in upper case and unsafe bases are outputted in lower case.
Optimized version of the algorithm.
Output now indicates on which gaps search was aborted because of the memory limit.
Reorganized parallel execution.
Proper synchronization for access to the memuse hash table.
Switched to GATB 1.0.5.
The maximum memory limitation option is now total for all threads. This is then divided evenly to all threads.
Memory usage tracking now includes all major data structures excluding the DBG.
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