# install minimap2
git clone https://github.com/lh3/minimap2
cd minimap2 && make
# install the k8 javascript shell
curl -L https://github.com/attractivechaos/k8/releases/download/v0.2.4/k8-0.2.4.tar.bz2 | tar -jxf -
cp k8-0.2.4/k8-`uname -s` k8 # or copy it to a directory on your $PATH
# export PATH="$PATH:`pwd`:`pwd`/misc" # run this if k8, minimap2 or paftools.js not on your $PATH
minimap2 --cs test/MT-human.fa test/MT-orang.fa | paftools.js view - # view alignment
minimap2 -c test/MT-human.fa test/MT-orang.fa | paftools.js stat - # basic alignment statistics
minimap2 -c --cs test/MT-human.fa test/MT-orang.fa \
| sort -k6,6 -k8,8n | paftools.js call -L15000 - # calling variants from asm-to-ref alignment
minimap2 -c test/MT-human.fa test/MT-orang.fa \
| paftools.js liftover -l10000 - <(echo -e "MT_orang\t2000\t5000") # liftOver
# no test data for the following examples
paftools.js junceval -e anno.gtf splice.sam > out.txt # compare splice junctions to annotations
paftools.js splice2bed splice.sam > splice.bed # convert PAF/SAM to BED12
paftools.js gff2bed anno.gtf > anno.bed # convert GTF/GFF3 to BED12paftools.js is a script that processes alignments in the PAF format, such as converting between formats, evaluating mapping accuracy, lifting over BED files based on alignment, and calling variants from assembly-to-assembly alignment. This script requires the k8 Javascript shell to run. On Linux or Mac, you can download the precompiled k8 binary with:
curl -L https://github.com/attractivechaos/k8/releases/download/v0.2.4/k8-0.2.4.tar.bz2 | tar -jxf -
cp k8-0.2.4/k8-`uname -s` $HOME/bin/k8 # assuming $HOME/bin in your $PATHIt is highly recommended to copy the executable k8 to a directory on your
$PATH such as /usr/bin/env can find it. Like python scripts, once you
install k8, you can launch paftools.js in one of the two ways:
path/to/paftools.js # only if k8 is on your $PATH
k8 path/to/paftools.jsIn a nutshell, paftools.js has the following commands:
Usage: paftools.js <command> [arguments]
Commands:
view convert PAF to BLAST-like (for eyeballing) or MAF
splice2bed convert spliced alignment in PAF/SAM to BED12
sam2paf convert SAM to PAF
delta2paf convert MUMmer's delta to PAF
gff2bed convert GTF/GFF3 to BED12
stat collect basic mapping information in PAF/SAM
liftover simplistic liftOver
call call variants from asm-to-ref alignment with the cs tag
bedcov compute the number of bases covered
mapeval evaluate mapping accuracy using mason2/PBSIM-simulated FASTQ
mason2fq convert mason2-simulated SAM to FASTQ
pbsim2fq convert PBSIM-simulated MAF to FASTQ
junceval evaluate splice junction consistency with known annotations
ov-eval evaluate read overlap sensitivity using read-to-ref mapping
paftools.js seamlessly reads both plain text files and gzip'd text files.
The pbsim2fq command of paftools.js converts the MAF output of pbsim
to FASTQ and encodes the true mapping position in the read name in a format like
S1_33!chr1!225258409!225267761!-. Similarly, the mason2fq command
converts mason2 simulated SAM to FASTQ.
Command mapeval evaluates mapped SAM/PAF. Here is example output:
Q 60 32478 0 0.000000000 32478
Q 22 16 1 0.000030775 32494
Q 21 43 1 0.000061468 32537
Q 19 73 1 0.000091996 32610
Q 14 66 1 0.000122414 32676
Q 10 27 3 0.000214048 32703
Q 8 14 1 0.000244521 32717
Q 7 13 2 0.000305530 32730
Q 6 46 1 0.000335611 32776
Q 3 10 1 0.000366010 32786
Q 2 20 2 0.000426751 32806
Q 1 248 94 0.003267381 33054
Q 0 31 17 0.003778147 33085
U 3
where each Q-line gives the quality threshold, the number of reads mapped with mapping quality equal to or greater than the threshold, number of wrong mappings, accumulative mapping error rate and the accumulative number of mapped reads. The U-line, if present, gives the number of unmapped reads if they are present in the SAM file.
Suppose the reported mapping coordinate overlap with the true coordinate like the following:
truth: --------------------
mapper: ----------------------
|<- l1 ->|<-- o -->|<-- l2 -->|
Let r=o/(l1+o+l2). The reported mapping is considered correct if r>0.1 by
default.
Command ov-eval takes sorted read-to-reference alignment and read overlaps in PAF as input, and evaluates the sensitivity. For example:
minimap2 -cx map-pb ref.fa reads.fq.gz | sort -k6,6 -k8,8n > reads-to-ref.paf
minimap2 -x ava-pb reads.fq.gz reads.fq.gz > ovlp.paf
k8 ov-eval.js reads-to-ref.paf ovlp.pafThe call command of paftools.js calls variants from coordinate-sorted assembly-to-reference alignment. It calls variants from the cs tag and identifies confident/callable regions as those covered by exactly one contig. Here are example command lines:
minimap2 -cx asm5 -t8 --cs ref.fa asm.fa > asm.paf # keeping this file is recommended; --cs required!
sort -k6,6 -k8,8n asm.paf > asm.srt.paf # sort by reference start coordinate
k8 paftools.js call asm.srt.paf > asm.var.txtHere is sample output:
V chr1 2276040 2276041 1 60 c g LJII01000171.1 1217409 1217410 +
V chr1 2280409 2280410 1 60 a g LJII01000171.1 1221778 1221779 +
V chr1 2280504 2280505 1 60 a g LJII01000171.1 1221873 1221874 +
R chr1 2325140 2436340
V chr1 2325287 2325287 1 60 - ct LJII01000171.1 1272894 1272896 +
V chr1 2325642 2325644 1 60 tt - LJII01000171.1 1273251 1273251 +
V chr1 2326051 2326052 1 60 c t LJII01000171.1 1273658 1273659 +
V chr1 2326287 2326288 1 60 c t LJII01000171.1 1273894 1273895 +
where a line starting with R gives regions covered by one query contig, and a
V-line encodes a variant in the following format: chr, start, end, query depth,
mapping quality, REF allele, ALT allele, query name, query start, end and the
query orientation. Generally, you should only look at variants where column 5
is one.
By default, when calling variants, "paftools.js call" ignores alignments 50kb or shorter; when deriving callable regions, it ignores alignments 10kb or shorter. It uses two thresholds to avoid edge effects. These defaults are designed for long-read assemblies. For short reads, both should be reduced.