Workflow Examples

Isaac Turner edited this page May 28, 2018 · 44 revisions

Table of Contents

Automatically Running McCortex Pipeline

This is the fastest way to use McCortex to go from sequence files to VCFs. It runs with the most common settings and you only need to run two commands. You need to create a text file listing your samples and their input sequences files (any mix of SAM/BAM/FASTA/FASTQ or gzipped FASTA/FASTQ is ok). Then give this file to scripts/make-pipeline.pl, along with a reference if you have one. Read more about the McCortex Pipeline. Otherwise the sections below will walk you through the individual steps to use McCortex.

Graph Construction

Construct graphs for three samples (using 70GB ram):

mccortex31 build -m 70G -k 31 --sample NA12878 --seq input.fq.gz NA12878.ctx
mccortex31 build -m 70G -k 31 --sample Mickey --seq2 reads.1.fq.gz:reads.2.fq.gz Mickey.ctx
mccortex31 build -m 70G -k 31 --sample Minnie --seq data.bam Minnie.ctx

Construct graph for the reference (hg19)

mccortex31 build -m 70G -k 31 --sample hg19 --seq hg19.fa.gz hg19.ctx

Graphical Fragment Assembly (GFA) representaiton

Generate a GFA representation of the unitig graph with:

mccortex31 unitigs --gfa graph.ctx > graph.gfa

Merging Graphs

Merge multiple graphs together. Graphs are kept as separate colours unless the --flatten option is used. To combine three graphs into refAndSamples.ctx:

mccortex31 join --out refAndSamples.ctx NA12878.ctx Mickey.ctx Minnie.ctx

Colour kmer distance matrix

To generate a matrix of how many kmers are shared with between any pair of colours, use the dist command:

mccortex31 dist -m 50G --out dist.tsv NA12878.ctx Mickey.ctx Minnie.ctx

Generated file dist.tsv is a tab separated matrix like so:

. col0  col1
col0  2874186023  2107456247
col1  0 2796104371

Kmer Error Cleaning

There are two options here depending on sequence coverage.

A) 'Clean' graphs to remove sequencing error (per sample, for high coverage samples):

mccortex31 clean -m 60G --out NA12878.clean.ctx NA12878.ctx
mccortex31 clean -m 60G --out Mickey.clean.ctx Mickey.ctx
mccortex31 clean -m 60G --out Minnie.clean.ctx Minnie.ctx

...then merge graphs into file refAndSamples.ctx. Uses 80GB ram:

mccortex31 join -m 80G --out refAndSamples.clean.ctx hg19.ctx NA12878.clean.ctx Mickey.clean.ctx Minnie.clean.ctx

B) Alternatively merge uncleaned samples then clean on the population (multiple low depth samples). Uses 100GB ram:

mccortex31 clean -m 100G --out samples.clean.ctx NA12878.ctx Mickey.ctx Minnie.ctx
mccortex31 join -m 80G --out refAndSamples.ctx hg19.ctx samples.clean.ctx

Now we have cortex graphs of the reference and our samples with sequencing error removed.

C) Another way to achieve pooled cleaning is to create a graph of kmers you want to keep, and intersect every sample with it. Note 0: loads each sample into colour 0:

mccortex31 clean -m 100G --out keep.kmers.ctx 0:NA12878.ctx 0:Mickey.ctx 0:Minnie.ctx
mccortex31 join -m 100G --out pop.clean.ctx --intersect keep.kmers.ctx NA12878.ctx Mickey.ctx Minnie.ctx

Plot Coverage Distribution

The command mccortex31 clean has the option of writing out histograms of supernode lengths and coverages with the --covg-before, --covg-after, --len-before and --len-after arguments. You can plot the results of these using a pair of scripts bundled with McCortex in the scripts/ directory (requires R and R packages ggplot2 and gridExtra):

scripts/R/plot-covg-hist.R covg.before_cleaning.csv covg.before_cleaning.pdf
scripts/R/plot-covg-hist.R covg.after_cleaning.csv covg.after_cleaning.pdf
scripts/R/plot-length-hist.R length.before_cleaning.csv length.before_cleaning.pdf
scripts/R/plot-length-hist.R length.after_cleaning.csv length.after_cleaning.pdf

Read Threading

Read threading aligns reads against the graph and generates graph `links' which store long range connectivity information. This is per-sample information (each sample has its own set of links).

Before threading reads through the graph, you must run inferedges:

mccortex31 inferedges -m 50G in.ctx

This edits in.ctx to add missing edges between kmers. Later steps assume all edges between adjacent kmers already exist.

Before threading reads, paired end reads that overlap should be merged. There are many good read merging programs that can be used, read more on the read threading page.

Thread reads through the graph to assist in assembly, generating file refAndSamples.ctp.gz:

mccortex31 thread -m 80G --seq2 NA12878.1.fq:NA12878.2.fq --out NA12878.ctp.gz refAndSamples.ctx:1
mccortex31 thread -m 80G --seqi Mickey.bam --out Mickey.ctp.gz refAndSamples.ctx:2
mccortex31 thread -m 80G --seqi Minnie.sam --out Minnie.ctp.gz refAndSamples.ctx:3

Now the link files NA12878.ctp.gz, Mickey.ctp.gz and Minnie.ctp.gz are ready to be used alongside their respective graphs, e.g.:

mccortex31 contigs -m 100G -p 1:NA12878.ctp.gz -p 2:Mickey.ctp.gz -p 3:Minnie.ctp.gz refAndSamples.ctx

The reference is colour 0 in the above example. Read more on graph links.

Link Error Cleaning

Once a set of links have been generated, we need to remove links due to sequencing error. This is done in two steps: first we use the first 1,000 kmers (with links) to pick a cleaning threshold, then we use the cleaning threshold to create a cleaned set of links.

mccortex31 links -T link.stats.txt -L 1000 graph.raw.ctp.gz
LINK_THRESH=`grep 'suggested_cutoff=' link.stats.txt | grep -oE '[0-9,]+$'`
mccortex31 links --clean $LINK_THRESH --out graph.clean.ctp.gz graph.raw.ctp.gz

Cleaning links also removes redundant links. Combined with removing rare links, this reduces the memory required to store graph connectivity.

Merge Link Files

Similar to the join command, pjoin merges link files.

mccortex31 pjoin -m 30G -o refAndNA12878.ctp.gz ref.ctp.gz NA12878.ctp.gz

Calling variants

There are two algorithms for identifying putative variants in a McCortex graph -- the bubble and breakpoint callers. Both work with one or more graph colours (i.e. multi-sample). The bubble caller optionally takes a reference colour, the breakpoint caller requires a reference.

Calling variants is a four step process:

  1. make graph calls with breakpoints or bubbles command
  2. convert calls to VCF with calls2vcf command
  3. sort VCFs, merge and remove duplicates, compress and index etc.
  4. add sample coverage with vcfcov command
  5. genotype samples with vcfgeno command

Bubble caller

mccortex31 bubbles -m 100G --out denovo.bubbles.gz --haploid 0 -p refAndSamples.ctp.gz refAndSamples.ctx

Uses the graph and links (refAndSamples.ctx, refAndSamples.ctp.gz) to call variants that are saved to the file denovo.bubbles.gz. We also specify that the first colour is a haploid sample (with --haploid 0). This is useful in removing bubbles caused by repeats rather than real variants.

Next we extract the 5' flanks from the bubble file:

./scripts/cortex_print_flanks.sh denovo.bubbles.gz > denovo.5pflank.fa

Creates the file denovo.5pflank.fa. We then map the putative variant flanks to our reference (in this case hg19). We can do this with stampy:

stampy.py -G hg19 hg19.fa  # create hg19.stidx
stampy.py -g hg19 -H hg19  # create hg19.sthash
stampy.py -g hg19 -h hg19 --inputformat=fasta -M denovo.5pflank.fa > denovo.5pflank.sam

or BWA:

bwa index hg19.fa
bwa mem hg19.fa denovo.5pflank.fa > denovo.5pflank.sam

Then we align the alleles to the reference and generate a VCF

mccortex31 calls2vcf -F denovo.5pflank.sam -o denovo.vcf bubbles.txt.gz hg19.fa

This generates a VCF of sites without genotypes and will contain duplicates. See the VCF post processing section to sort, remove duplicates, index and clean up your VCF.

Breakpoint caller

If you have an assembled genome, you can compare your sequenced samples to it to identify large variants and re-arrangements:

mccortex31 breakpoints -m 100G --seq hg19.fa --out breakpoints.txt.gz -p PlutoAndGoofy.ctp.gz PlutoAndGoofy.ctx

The breakpoints command identifies where your samples diverge from the reference and rejoin it elsewhere. This could be small regions of clustered SNPs, indels, inversions or translocations.

We can generate a VCF of small events using calls2vcf:

mccortex31 calls2vcf -o breakpoints.vcf breakpoints.txt.gz hg19.fa

This generates a VCF of sites without genotypes and will contain duplicates. See the VCF post processing section to sort, remove duplicates, index and clean up your VCF.

Results can be plotted on a Circos plot using a perl script in scripts/:

gzip -dc breakpoints.txt.gz | scripts/make-circos.pl out-dir -
cd out-dir
circos

Plot appears in out-dir/circos.png.

Post processing a VCF

Once you have called variants with the Bubble Caller or the Breakpoint Caller, you are left with an unsorted VCF calls.raw.vcf. Our proposed post-processing sorts, removes duplicates, renames variants, compresses and indexes the VCF:

./scripts/bash/vcf-sort calls.raw.vcf > calls.sorted.vcf
bcftools norm --remove-duplicates --fasta-ref ref.fa --multiallelics -any $< | \
  ./scripts/bash/vcf-rename > calls.norm.vcf
bgzip calls.norm.vcf
bcftools index calls.norm.vcf.gz

Next see VCF coverage and genotyping.

Annotating a VCF with graph coverage

Given a sorted VCF, a reference and a cortex graph, we can annotate the VCF with the coverage for each sample in the graph:

mccortex31 vcfcov -m 60G --out out.vcf.gz --ref ref.fa in.vcf graph.ctx

Note: the VCF must be sorted and the reference must contain all the chromosomes in the VCF. The order of the chromosomes in the VCF and reference do not matter and do not need to be the same.

For each sample in the graph, we add a sample to the VCF with the same name. For each sample we add two fields:

  • K31R mean coverage of kmers unique to the reference
  • K31A mean coverage of kmers unique to the alternative allele

vcfcov works with biallelic and multiallelic VCFs. The VCF must be sorted and will be printed in the order it was read in. With no --out argument vcfcov prints to STDOUT.

Coverage is calculated by assembling all local haplotypes and finding which kmers uniquely support an alternative allele. We then report the number and mean kmer coverage on each reference and alternative allele.

See full page on VCF Genotyping.

Genotyping

After adding sample coverage for variants using the vcfcov -- one sample at a time or all at once -- you can genotype a VCF with:

mccortex31 vcfgeno -m 60G --out out.vcf.gz --cov 40 in.vcf graph.ctx

Adding -r (--rm-cov) will remove the annotations added by vcfcov after setting the genotype.

See full page on VCF Genotyping.

Assemble Contigs

Pull out contigs from an individual in a population. Fills in low coverage gaps in sequence from the population if unconfounded.

mccortex31 contigs -p refAndSamples.ctp.gz --colour 1 --out refAndSamples.contigs.fa refAndSamples.ctx

The contigs command uses random kmers to seed contigs, therefore it may print duplicate contigs and some contigs that are substrings of others. To remove duplicate contigs you can use the rmsubstr command:

mccortex31 rmsubstr -m 10G --out refAndSamples.contigs.rmdup.fa refAndSamples.contigs.fa

More on the contig assembly page.

Filter A Subgraph

Pull out a region of interest using a set of reads or kmers. The subgraph command searches out from the seed kmers by dist kmers (dist >= 0).

mccortex31 subgraph --seq contig.fa contig.ctx 100 graph.ctx

Input is graph.ctx, output is saved to contig.ctx. You can specify particular colours on the input to keep in the output. Add the --invert option to invert the kmers selected - only take those further than dist kmers.

Filter Reads Against A Graph

Filter reads by whether or not they share a kmer with a graph:

mccortex31 reads [--fasta|--fastq] --seq in1.fa out.se --seq2 in.1.fq:in.2.fq out.pair in.ctx

Will save reads from in1.fa to out.se.fa.gz and from in.1.fq,in.2.fq to out.pair.1.fa.gz,out.pair.2.fa.gz. Output options are --fasta or --fastq. Paired end reads (--seq2) are considered touching the graph if either read shares one or more kmers with the graph. Add the --invert option to select reads that DO NOT touch the graph.

Plot Graph

Requires GraphViz which provides the dot command. Flattens any colours in the graph:

./scripts/perl/mccortex-graph-to-graphviz.pl graph.ctx:0,3 | dot -Tpdf > kmers.pdf

Or to merge adjacent kmers add the --simplify option:

./scripts/perl/mccortex-graph-to-graphviz.pl --simplify graph.ctx:0,3 | dot -Tpdf > contigs.pdf

Or you can just use ./scripts/perl/mccortex-graph-to-graphviz.pl to generate dot files.

You can generate a plot of a de Bruijn graph straight from sequence files using scripts/seq2pdf.sh:

./scripts/seq2pdf.sh --simplify 3 <(echo ACAACACGT) <(echo CCACACAA) > out.pdf

Usage is: ./scripts/seq2pdf.sh [--simplify] <kmer> <file1> [file2] > out.pdf. Files can be of types FASTA, FASTQ, SAM, BAM, TXT or even gzipped. Requires graphviz.

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