Package: tigmint
Correct misassemblies using linked readsCut sequences at positions with few spanning molecules.Written byShaun Jackman, Lauren Coombe, and Justin Chu.bioRxiv doi:10.1101/304253·Slides·PosterDescriptionTigmint 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.InstallationInstall Tigmint using BrewInstallLinuxbrewon Linux or Windows Subsystem for Linux (WSL), or
installHomebrewon macOS, and then run the commandbrew install tigmintInstall Tigmint from the source codeDownload and extract the source code. Compiling is not needed.git clone https://github.com/bcgsc/tigmint && cd tigmintorcurl -L https://github.com/bcgsc/tigmint/archive/master.tar.gz | tar xz && mv tigmint-master tigmint && cd tigmintDependenciesInstall Python package dependenciespip3installintervaltreepybedtoolspysamstatisticsTigmint uses Bedtools, BWA and Samtools. These dependencies may be installed usingHomebrewon macOS orLinuxbrewon Linux.Install the dependencies of TigmintbrewinstallbedtoolsbwasamtoolsInstall the dependencies of ARCS (optional)brewtapbrewsci/bio
brewinstallarcslinks-scaffolderInstall the dependencies for calculating assembly metrics (optional)brewinstallabyssseqtkUsageTo run Tigmint on the draft assemblydraft.fawith the readsreads.fq.gz, which have been run throughlongranger basic:samtoolsfaidxdraft.fa
bwaindexdraft.fa
bwamem-t8-p-Cdraft.fareads.fq.gz|samtoolssort-@8-tBX-odraft.reads.sortbx.bam
tigmint-moleculedraft.reads.sortbx.bam|sort-k1,1-k2,2n-k3,3n>draft.reads.molecule.bed
tigmint-cut-p8-odraft.tigmint.fadraft.fadraft.reads.molecule.bedbwa mem -Cis used to copy the BX tag from the FASTQ header to the SAM tags.samtools sort -tBXis used to sort first by barcode and then position.Alternatively, you can run the Tigmint pipeline using the Makefile driver scripttigmint-make. To run Tigmint on the draft assemblymyassembly.fawith the readsmyreads.fq.gz, which have been run throughlongranger basic:tigmint-maketigmintdraft=myassemblyreads=myreadsTo run both Tigmint and scaffold the corrected assembly withARCS:tigmint-makearcsdraft=myassemblyreads=myreadsTo run Tigmint, ARCS, and calculate assembly metrics using the reference genomeGRCh38.fa:tigmint-makemetricsdraft=myassemblyreads=myreadsref=GRCh38G=3088269832Notetigmint-makeis a Makefile script, and so anymakeoptions may also be used withtigmint-make, such as-n(--dry-run).The file extension of the assembly must be.faand the reads.fq.gz, and the extension is not included in the parametersdraftandreads. These specific file name requirements result from implementing the pipeline in GNU Make.tigmint-make commandstigmint: Run Tigmint, and produce a file named$draft.tigmint.faarcs: Run Tigmint and ARCS, and produce a file name$draft.tigmint.arcs.fametrics: Run, Tigmint, ARCS, and calculate assembly metrics usingabyss-facandabyss-samtobreak, and produce TSV files.Parameters of Tigmintdraft: Name of the draft assembly,draft.fareads: Name of the reads,reads.fq.gzspan=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 threadsParameters of ARCSc=5e=30000r=0.05Parameters of LINKSa=0.1l=10Parameters for calculating assembly metricsref: Reference genome,ref.fa, for calculating assembly contiguity metricsG: Size of the reference genome, for calculating NG50 and NGA50TipsIf 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-Coption to include the barcode in the BX tag of the alignments.Sort by BX tag usingsamtools sort -tBX.Merge multiple BAM files usingsamtools merge -tBX.SupportAfter first looking for existing issue athttps://github.com/bcgsc/tigmint/issues, please report a new issue athttps://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.Pipeline