- Introduction & Installation
- Mapping Genomic Reads
- Mapping Long RNA-seq Reads
- Full-Genome Alignment
- Read Overlap
This cookbook walks you through a variety of applications of minimap2 and its
companion script paftools.js
. All data here are freely available from the
minimap2 release page at version tag v2.10. Some examples only work
with v2.10 or later.
To acquire the data used in this cookbook and to install minimap2 and paftools, please follow the command lines below:
# install minimap2 executables
curl -L https://github.com/lh3/minimap2/releases/download/v2.28/minimap2-2.28_x64-linux.tar.bz2 | tar jxf -
cp minimap2-2.28_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/v2.10/cookbook-data.tgz | tar zxf -
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.
minimap2 -ax sr -t4 ecoli_ref.fa ecoli_mason_1.fq ecoli_mason_2.fq > mapped-sr.sam
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:
- Mapping quality (mapQ) threshold
- Number of mapped reads between this threshold and the previous mapQ threshold.
- Number of wrong mappings in the same mapQ interval
- Accumulative mapping error rate
- 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 pbsim2 and mason2, we
provide scripts to generate the right format. Simulated reads in this cookbook
were created with the following command lines:
# in the pbsim2 source code directory:
src/pbsim --depth 1 --length-min 5000 --length-mean 20000 --accuracy-mean 0.95 --hmm_model data/R94.model ../ecoli_ref.fa
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 -2 > ecoli_mason_2.fq
minimap2 -ax splice SIRV_E2.fa SIRV_ont-cdna.fa > aln.sam
You can compare the alignment to the true annotations with:
paftools.js junceval SIRV_E2C.gtf aln.sam
It gives the percentage of introns found in the annotation. For SIRV data, it is possible to achieve higher junction accuracy with
minimap2 -ax splice --splice-flank=no SIRV_E2.fa SIRV_ont-cdna.fa | paftools.js junceval SIRV_E2C.gtf
This is because minimap2 models one additional evolutionarily conserved base
around a canonical junction, but SIRV doesn't honor this signal. Option
--splice-flank=no
asks minimap2 no to model this additional base.
In the output a tag ts:A:+
indicates that the read strand is the same as the
transcript strand; ts:A:-
indicates the read strand is opposite to the
transcript strand. This tag is inferred from the GT-AG signal and is thus only
available to spliced reads.
minimap2 -ax splice -k14 -uf SIRV_E2.fa SIRV_ont-drna.fa > aln.sam
Direct-RNA reads are noisier, so we use a shorter k-mer for improved
sensitivity. Here, option -uf
forces minimap2 to map reads to the forward
transcript strand only because direct-RNA reads are stranded. Again, applying
--splice-flank=no
helps junction accuracy for SIRV data.
minimap2 -ax splice -uf -C5 SIRV_E2.fa SIRV_iso-seq.fq > aln.sam
Option -C5
reduces the penalty on non-canonical splicing sites. It helps
to align such sites correctly for data with low error rate such as Iso-seq
reads and traditional cDNAs. On this example, minimap2 makes one junction
error. Applying --splice-flank=no
fixes this alignment error.
Note that the command line above is optimized for the final Iso-seq reads.
PacBio's Iso-seq pipeline produces intermediate sequences at varying quality.
For example, some intermediate reads are not stranded. For these reads, option
-uf
will lead to more errors. Please revise the minimap2 command line
accordingly.
# option "--cs" is recommended as paftools.js may need it
minimap2 -cx asm5 --cs ecoli_ref.fa ecoli_canu.fa > ecoli_canu.paf
Here ecoli_canu.fa
is the Canu assembly of ecoli_p6_25x_canu.fa
. This
command line outputs alignments in the PAF format. Use -a
instead of
-c
to get output in the SAM format.
minimap2 -cx asm20 --cs ecoli_ref.fa ecoli_O104:H4.fa > ecoli_O104:H4.paf
sort -k6,6 -k8,8n ecoli_O104:H4.paf | paftools.js call -f ecoli_ref.fa -L10000 -l1000 - > out.vcf
Minimap2 has three presets for full-genome alignment: "asm5" for sequence divergence below 1%, "asm10" for divergence around a couple of percent and "asm20" for divergence not more than 10%. In theory, with the right setting, minimap2 should work for sequence pairs with sequence divergence up to ~15%, but this has not been carefully evaluated.
# option "--cs" required; minimap2-r741 or higher required for the "asm20" preset
minimap2 -cx asm20 --cs ecoli_ref.fa ecoli_O104:H4.fa | paftools.js view - | less -S
This prints the alignment in a BLAST-like format.
# don't forget the "--cs" option; otherwise it doesn't work
minimap2 -cx asm5 --cs ecoli_ref.fa ecoli_canu.fa \
| sort -k6,6 -k8,8n \
| paftools.js call -f ecoli_ref.fa - > out.vcf
Without option -f
, paftools.js call
outputs in a custom format. In this
format, lines starting with R
give the regions covered by one contig only.
This information is not available in the VCF output.
minimap2 -DP -k19 -w19 -m200 ecoli_ref.fa ecoli_ref.fa > out.paf
Option -D
asks minimap2 to ignore anchors from perfect self match and -P
outputs all chains. For large nomes, we don't recommend to perform base-level
alignment (with -c
, -a
or --cs
) when -P
is applied. This is because
base-alignment is slow and occasionally gives wrong alignments close to the
diagonal of a dotter plot. For E. coli, though, base-alignment is still fast.
minimap2 -cx asm5 --cs ecoli_ref.fa ecoli_canu.fa > ecoli_canu.paf
echo -e 'tig00000001\t200000\t300000' | paftools.js liftover ecoli_canu.paf -
This lifts over a region on query sequences to one or multiple regions on reference sequences. Note that this paftools.js command may not be efficient enough to lift millions of regions.
# 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
# If you have miniasm installed:
miniasm -f ecoli_p6_25x_canu.fa overlap.paf > asm.gfa
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.
# 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
).