Correct misassemblies using linked reads
Project description
Correct misassemblies using linked reads
Cut sequences at positions with few spanning molecules.
Written by Shaun Jackman, Lauren Coombe, and Justin Chu.
bioRxiv doi:10.1101/304253 · Slides · Poster
Description
Tigmint identifies and corrects misassemblies using linked reads from 10x Genomics Chromium. The reads are first aligned to the assembly, and the extents of the large DNA molecules are inferred from the alignments of the reads. The physical coverage of the large molecules is more consistent and less prone to coverage dropouts than that of the short read sequencing data. The sequences are cut at positions that have insufficient spanning molecules. Tigmint outputs a BED file of these cut points, and a FASTA file of the cut sequences.
Each window of a specified fixed size is checked for a minimum number of spanning molecules. Sequences are cut at those positions where a window with sufficient coverage is followed by some number of windows with insufficient coverage is then followed again by a window with sufficient coverage.
Installation
Install Tigmint using Brew
Install Linuxbrew on Linux or Windows Subsystem for Linux (WSL), or install Homebrew on macOS, and then run the command
brew install tigmint
Install Tigmint from the source code
Download and extract the source code. Compiling is not needed.
git clone https://github.com/bcgsc/tigmint && cd tigmint
or
curl -L https://github.com/bcgsc/tigmint/archive/master.tar.gz | tar xz && mv tigmint-master tigmint && cd tigmint
Dependencies
Install Python package dependencies
pip3 install intervaltree pybedtools pysam statistics
Tigmint uses Bedtools, BWA and Samtools. These dependencies may be installed using Homebrew on macOS or Linuxbrew on Linux.
Install the dependencies of Tigmint
brew install bedtools bwa samtools
Install the dependencies of ARCS (optional)
brew tap brewsci/bio
brew install arcs links-scaffolder
Install the dependencies for calculating assembly metrics (optional)
brew install abyss seqtk
Usage
To run Tigmint on the draft assembly draft.fa
with the reads reads.fq.gz
, which have been run through longranger basic
:
samtools faidx draft.fa
bwa index draft.fa
bwa mem -t8 -p -C draft.fa reads.fq.gz | samtools sort -@8 -tBX -o draft.reads.sortbx.bam
tigmint-molecule draft.reads.sortbx.bam | sort -k1,1 -k2,2n -k3,3n >draft.reads.molecule.bed
tigmint-cut -p8 -o draft.tigmint.fa draft.fa draft.reads.molecule.bed
bwa mem -C
is used to copy the BX tag from the FASTQ header to the SAM tags.samtools sort -tBX
is used to sort first by barcode and then position.
Alternatively, you can run the Tigmint pipeline using the Makefile driver script tigmint-make
. To run Tigmint on the draft assembly myassembly.fa
with the reads myreads.fq.gz
, which have been run through longranger basic
:
tigmint-make tigmint draft=myassembly reads=myreads
To run both Tigmint and scaffold the corrected assembly with ARCS:
tigmint-make arcs draft=myassembly reads=myreads
To run Tigmint, ARCS, and calculate assembly metrics using the reference genome GRCh38.fa
:
tigmint-make metrics draft=myassembly reads=myreads ref=GRCh38 G=3088269832
Note
tigmint-make
is a Makefile script, and so anymake
options may also be used withtigmint-make
, such as-n
(--dry-run
).- The file extension of the assembly must be
.fa
and the reads.fq.gz
, and the extension is not included in the parametersdraft
andreads
. These specific file name requirements result from implementing the pipeline in GNU Make.
tigmint-make commands
tigmint
: Run Tigmint, and produce a file named$draft.tigmint.fa
arcs
: Run Tigmint and ARCS, and produce a file name$draft.tigmint.arcs.fa
metrics
: Run, Tigmint, ARCS, and calculate assembly metrics usingabyss-fac
andabyss-samtobreak
, and produce TSV files.
Parameters of Tigmint
draft
: Name of the draft assembly,draft.fa
reads
: Name of the reads,reads.fq.gz
span=20
: Number of spanning molecules thresholdwindow=1000
: Window size (bp) for checking spanning moleculesminsize=2000
: Minimum molecule sizeas=0.65
: Minimum AS/read length rationm=5
: Maximum number of mismatchesdist=50000
: Maximum distance (bp) between reads to be considered the same moleculemapq=0
: Mapping quality thresholdtrim=0
: Number of bases to trim off contigs following cutst=8
: Number of threads
Parameters of ARCS
c=5
e=30000
r=0.05
Parameters of LINKS
a=0.1
l=10
Parameters for calculating assembly metrics
ref
: Reference genome,ref.fa
, for calculating assembly contiguity metricsG
: Size of the reference genome, for calculating NG50 and NGA50
Tips
- If your barcoded reads are in multiple FASTQ files, the initial alignments of the barcoded reads to the draft assembly can be done in parallel and merged prior to running Tigmint.
- When aligning with BWA-MEM, use the
-C
option to include the barcode in the BX tag of the alignments. - Sort by BX tag using
samtools sort -tBX
. - Merge multiple BAM files using
samtools merge -tBX
.
Support
After first looking for existing issue at https://github.com/bcgsc/tigmint/issues, please report a new issue at https://github.com/bcgsc/tigmint/issues/new. Please report the names of your input files, the exact command line that you are using, and the entire output of Tigmint.
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