minimap2/cookbook.md

Table of Contents

• Installation
• Mapping Genomic Reads
  • Mapping long reads
  • Mapping Illumina paired-end reads
  • Evaluating mapping accuracy with simulated reads (for developers)
• Read Overlap
  • Long-read overlap
  • Evaluating overlap sensitivity (for developers)

Installation

# install minimap2 executables
curl -L https://github.com/lh3/minimap2/releases/download/v2.9/minimap2-2.9_x64-linux.tar.bz2 | tar jxf -
cp minimap2-2.9_x64-linux/{minimap2,k8,paftools.js} .  # copy executables
export PATH="$PATH:"`pwd`                              # put the current directory on PATH
# download example datasets
curl -L https://github.com/lh3/minimap2/releases/download/v2.0/cookbook-data.tgz | tar zxf -

Mapping Genomic Reads

Mapping long reads

minimap2 -ax map-pb -t4 ecoli_ref.fa ecoli_p6_25x_canu.fa > mapped.sam

Alternatively, you can create a minimap2 index first and then map:

minimap2 -x map-pb -d ecoli-pb.mmi ecoli_ref.fa                      # create an index
minimap2 -ax map-pb ecoli-pb.mmi ecoli_p6_25x_canu.fa > mapped.sam

This will save you a couple of minutes when you map against the human genome. HOWEVER, key algorithm parameters such as the k-mer length and window size can't be changed after indexing. Minimap2 will give you a warning if parameters used in a pre-built index doesn't match parameters on the command line. Please always make sure you are using an intended pre-built index.

Mapping Illumina paired-end reads:

minimap2 -ax sr ecoli_ref.fa ecoli_mason_1.fq ecoli_mason_2.fq > mapped-sr.sam

Evaluating mapping accuracy with simulated reads (for developers)

minimap2 -ax sr ecoli_ref.fa ecoli_mason_1.fq ecoli_mason_2.fq | paftools.js mapeval -

The output is:

Q       60      19712   0       0.000000000     19712
Q       0       282     219     0.010953286     19994
U       6

where a U-line gives the number of unmapped reads (for SAM input only); a Q-line gives:

1. Mapping quality (mapQ) threshold
2. Number of mapped reads between this threshold and the previous mapQ threshold.
3. Number of wrong mappings in the same mapQ interval
4. Accumulative mapping error rate
5. Accumulative number of mappings

For paftools.js mapeval to work, you need to encode the true read positions in read names in the right format. For PBSIM and mason2, we provide scripts to generate the right format. Simulated reads in this cookbook were created with the following command lines:

# in PBSIM source code directory:
src/pbsim ../ecoli_ref.fa --depth 1 --sample-fastq sample/sample.fastq
paftools.js pbsim2fq ../ecoli_ref.fa.fai sd_0001.maf > ../ecoli_pbsim.fa

# mason2 simulation
mason_simulator --illumina-prob-mismatch-scale 2.5 -ir ecoli_ref.fa -n 10000 -o tmp-l.fq -or tmp-r.fq -oa tmp.sam
paftools.js mason2fq tmp.sam | seqtk seq -1 > ecoli_mason_1.fq
paftools.js mason2fq tmp.sam | seqtk seq -1 > ecoli_mason_2.fq

Read Overlap

Long read overlap

# For pacbio reads:
minimap2 -x ava-pb ecoli_p6_25x_canu.fa ecoli_p6_25x_canu.fa > overlap.paf
# For Nanopore reads (ava-ont also works with PacBio but not as good):
minimap2 -x ava-ont -r 10000 ecoli_p6_25x_canu.fa ecoli_p6_25x_canu.fa > overlap.paf

Here we explicitly applied -r 10000. We are considering to set this as the default for the ava-ont mode as this seems to improve the contiguity for nanopore read assembly (Loman, personal communication).

Minimap2 doesn't work well with short-read overlap.

Evaluating overlap sensitivity (for developers)

# read to reference mapping
minimap2 -cx map-pb ecoli_ref.fa ecoli_p6_25x_canu.fa > to-ref.paf
# evaluate overlap sensitivity
sort -k6,6 -k8,8n to-ref.paf | paftools.js ov-eval - overlap.paf

You can see that for PacBio reads, minimap2 achieves higher overlap sensitivity with -x ava-pb (99% vs 93% with -x ava-ont).

Mapping Long RNA-seq Reads

(Unfinished section. Data to come later...)

Mapping Nanopore direct-RNA reads

minimap2 -ax splice -k14 -uf ref.fa reads.fa > aln.sam