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Minimal but speedy quality control for nanopore reads in Rust 🐻

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nanoq

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Ultra-fast quality control and summary reports for nanopore reads

Overview

v0.8.5

Purpose

As sequencing throughput increases, so does the time for read summaries and basic quality control. Nanoq implements ultra-fast read filters and summary reports.

Citation

Nanoq is now published in JOSS. We would appreciate a citation if you are using nanoq for research. If you are using nanoq in industry, please see here for some suggestions how you could give back to the community 🙏

Steinig and Coin (2022). Nanoq: ultra-fast quality control for nanopore reads. Journal of Open Source Software, 7(69), 2991, https://doi.org/10.21105/joss.02991

Performance

Nanoq is as fast as seqtk-fqchk for summary statistics of small datasets (100,000 reads, + computes nanopore quality scores) and slightly faster on large datasets (3.5 million reads, 1.3x - 1.5x). In fast mode (no quality scores), nanoq is (~2-3x) faster than rust-bio-tools and seqkit stats for summary statistics and faster than other commonly used summary (up to 442x) and read filtering methods (up to 297x). Memory consumption is consistent and tends to be lower than other tools (~5-10x).

Tests

Nanoq comes with high test coverage for your peace of mind.

cargo test

Install

Cargo

cargo install nanoq

Conda

Explicit version (for some reason defaults to old version)

conda install -c conda-forge -c bioconda nanoq=0.8.5

Binaries

Precompiled binaries for Linux and MacOS are attached to the latest release.

VERSION=0.8.5
RELEASE=nanoq-${VERSION}-x86_64-unknown-linux-musl.tar.gz

wget https://github.com/esteinig/nanoq/releases/download/${VERSION}/${RELEASE}
tar xf nanoq-${VERSION}-x86_64-unknown-linux-musl.tar.gz

nanoq-${VERSION}-x86_64-unknown-linux-musl/nanoq -h

Usage

Nanoq accepts a file (-i) or stream (stdin) of reads in fast{a,q}.{gz,bz2,xz} format and outputs reads to file (-o) or stream (stdout).

nanoq -i test.fq.gz -o reads.fq
cat test.fq.gz | nanoq > reads.fq

Output compression is inferred from file extensions (gz, bz2, lzma).

nanoq -i test.fq -o reads.fq.gz

Output compression can be specified manually with -O and -c.

nanoq -i test.fq -O g -c 9 -o reads.fq.cmp

Reads can be filtered by minimum read length (-l), maximum read length (-m), minimum average read quality (-q) or maximum average read quality (-w).

nanoq -i test.fq -l 1000 -m 10000 -q 10 -w 15 > reads.fq

Read summaries without output can be obtained by directing to /dev/null or using the stats flag (-s):

nanoq -i test.fq > /dev/null
nanoq -i test.fq -s

Read qualities may be excluded from filters and statistics to speed up read iteration (-f).

nanoq -i test.fq.gz -f -s

Nanoq can be used to check on active sequencing runs and barcoded samples.

find /data/nanopore/run -name *.fastq -print0 | xargs -0 cat | nanoq -s
for i in {01..12}; do
  find /data/nanopore/run -name barcode${i}.fastq -print0 | xargs -0 cat | nanoq -s
done

Parameters

nanoq 0.8.5

Read filters and summary reports for nanopore data

USAGE:
    nanoq [FLAGS] [OPTIONS]

FLAGS:
    -f, --fast       Ignore quality values if present
    -h, --help       Prints help information
    -s, --stats      Summary statistics report
    -V, --version    Prints version information
    -v, --verbose    Verbose output statistics [multiple up to -vvv]

OPTIONS:
    -c, --compress-level <1-9>     Compression level to use if compressing output [default: 6]
    -i, --input <input>            Fast{a,q}.{gz,xz,bz}, stdin if not present
    -m, --max-len <INT>            Maximum read length filter (bp) [default: 0]
    -w, --max-qual <FLOAT>         Maximum average read quality filter (Q) [default: 0]
    -l, --min-len <INT>            Minimum read length filter (bp) [default: 0]
    -q, --min-qual <FLOAT>         Minimum average read quality filter (Q) [default: 0]
    -o, --output <output>          Output filepath, stdout if not present
    -O, --output-type <u|b|g|l>    u: uncompressed; b: Bzip2; g: Gzip; l: Lzma
    -t, --top <INT>                Number of top reads in verbose summary [default: 5]

Output

A basic read summary is output to stderr:

100000 400398234 5154 44888 5 4003 3256 8.90 9.49
  • number of reads
  • number of base pairs
  • N50 read length
  • longest read
  • shorted reads
  • mean read length
  • median read length
  • mean read quality
  • median read quality

Extended summaries analogous to NanoStat can be obtained using multiple -v flags (up to -vvv), including the top (-t) read lengths and qualities:

nanoq -i test.fq -f -s -vv
Nanoq Read Summary
====================

Number of reads:      100000
Number of bases:      400398234
N50 read length:      5154
Longest read:         44888 
Shortest read:        5
Mean read length:     4003
Median read length:   3256 
Mean read quality:    NaN 
Median read quality:  NaN


Read length thresholds (bp)

> 200       99104             99.1%
> 500       96406             96.4%
> 1000      90837             90.8%
> 2000      73579             73.6%
> 5000      25515             25.5%
> 10000     4987              05.0%
> 30000     47                00.0%
> 50000     0                 00.0%
> 100000    0                 00.0%
> 1000000   0                 00.0%


Top ranking read lengths (bp)

1. 44888       
2. 40044       
3. 37441       
4. 36543       
5. 35630

Benchmarks

Benchmarks evaluate processing speed and memory consumption of a basic read length filter and summary statistics on the even Zymo mock community (GridION) with comparisons to rust-bio-tools, seqtk fqchk, seqkit stats, NanoFilt, NanoStat and Filtlong. Time to completion and maximum memory consumption were measured using /usr/bin/time -f "%e %M", speedup is relative to the slowest command in the set. We note that summary statistics from rust-bio-tools and seqkit stats do not compute read quality scores and are therefore comparable to nanoq-fast.

Tasks:

  • stats: basic read set summaries
  • filter: minimum read length filter (into /dev/null)

Tools:

  • rust-bio-tools 0.28.0
  • nanostat 1.5.0
  • nanofilt 2.8.0
  • filtlong 0.2.1
  • seqtk 1.3-r126
  • seqkit 2.0.0
  • nanoq 0.8.2

Commands used for stats task:

  • nanostat (fq + fq.gz) --> NanoStat --fastq test.fq --threads 1
  • rust-bio (fq) --> rbt sequence-stats --fastq < test.fq
  • rust-bio (fq.gz) --> zcat test.fq.gz | rbt sequence-stats --fastq
  • seqtk-fqchk (fq + fq.gz) --> seqtk fqchk
  • seqkit stats (fq + fq.gz) --> seqkit stats -j1
  • nanoq (fq + fq.gz) --> nanoq --input test.fq --stats
  • nanoq-fast (fq + fq.gz) --> nanoq --input test.fq --stats --fast

Commands used for filter task:

  • filtlong (fq + fq.gz) --> filtlong --min_length 5000 test.fq > /dev/null
  • nanofilt (fq) --> NanoFilt --fastq test.fq --length 5000 > /dev/null
  • nanofilt (fq.gz) --> gunzip -c test.fq.gz | NanoFilt --length 5000 > /dev/null
  • nanoq (fq + fq.gz) --> nanoq --input test.fq --min-len 5000 > /dev/null
  • nanoq-fast (fq + fq.gz) --> nanoq --input test.fq --min-len 5000 --fast > /dev/null

Files:

  • zymo.fq: uncompressed (100,000 reads, ~400 Mbp)
  • zymo.fq.gz: compressed (100,000 reads, ~400 Mbp)
  • zymo.full.fq: uncompressed (3,491,078 reads, ~14 Gbp)

Data preparation:

wget "https://nanopore.s3.climb.ac.uk/Zymo-GridION-EVEN-BB-SN.fq.gz"
zcat Zymo-GridION-EVEN-BB-SN.fq.gz > zymo.full.fq
head -400000 zymo.full.fq > zymo.fq && gzip -k zymo.fq

Elapsed real time and maximum resident set size:

/usr/bin/time -f "%e %M"

Task and command execution:

Commands were run in replicates of 10 with a mounted benchmark data volume in the provided Docker container. An additional cold start iteration for each command was not considered in the final benchmarks.

for i in {1..11}; do
  for f in /data/*.fq; do 
    /usr/bin/time -f "%e %M" nanoq -f- s -i $f 2> benchmark
    tail -1 benchmark >> nanoq_stat_fq
  done
done

Benchmark results

Nanoq benchmarks on 3.5 million reads of the Zymo mock community (10 replicates)

stats + zymo.full.fq

command mb (sd) sec (sd) reads / sec speedup quality scores
nanostat 741.4 (0.09) 1260. (13.9) 2,770 01.00 x true
seqtk-fqchk 103.8 (0.04) 125.9 (0.15) 27,729 10.01 x true
seqkit-stats 18.68 (3.15) 125.3 (0.91) 27,861 10.05 x false
nanoq 35.83 (0.06) 94.51 (0.43) 36,938 13.34 x true
rust-bio 43.20 (0.08) 06.54 (0.05) 533,803 192.7 x false
nanoq-fast 22.18 (0.07) 02.85 (0.02) 1,224,939 442.1 x false

filter + zymo.full.fq

command mb (sd) sec (sd) reads / sec speedup
nanofilt 67.47 (0.13) 1160. (20.2) 3,009 01.00 x
filtlong 1516. (5.98) 420.6 (4.53) 8,360 02.78 x
nanoq 11.93 (0.06) 94.93 (0.45) 36,775 12.22 x
nanoq-fast 08.05 (0.05) 03.90 (0.30) 895,148 297.5 x

Nanoq benchmarks on 100,000 reads of the Zymo mock community (10 replicates)

stats + zymo.fq

command mb (sd) sec (sd) reads / sec speedup quality scores
nanostat 79.64 (0.14) 36.22 (0.27) 2,760 01.00 x true
nanoq 04.26 (0.09) 02.69 (0.02) 37,147 13.46 x true
seqtk-fqchk 53.01 (0.05) 02.28 (0.06) 43,859 15.89 x true
seqkit-stats 17.07 (3.03) 00.13 (0.00) 100,000 36.23 x false
rust-bio 16.61 (0.08) 00.22 (0.00) 100,000 36.23 x false
nanoq-fast 03.81 (0.05) 00.08 (0.00) 100,000 36.23 x false

stats + zymo.fq.gz

command mb (sd) sec (sd) reads / sec speedup quality scores
nanostat 79.46 (0.22) 40.98 (0.31) 2,440 01.00 x true
nanoq 04.44 (0.09) 05.74 (0.04) 17,421 07.14 x true
seqtk-fqchk 53.11 (0.05) 05.70 (0.08) 17,543 07.18 x true
rust-bio 01.59 (0.06) 05.06 (0.04) 19,762 08.09 x false
seqkit-stats 20.54 (0.41) 04.85 (0.02) 20,619 08.45 x false
nanoq-fast 03.95 (0.07) 03.15 (0.02) 31,746 13.01 x false

filter + zymo.fq

command mb (sd) sec (sd) reads / sec speedup
nanofilt 66.29 (0.15) 33.01 (0.24) 3,029 01.00 x
filtlong 274.5 (0.04) 08.49 (0.01) 11,778 03.89 x
nanoq 03.61 (0.04) 02.81 (0.28) 35,587 11.75 x
nanoq-fast 03.26 (0.06) 00.12 (0.01) 100,000 33.01 x

filter + zymo.fq.gz

command mb (sd) sec (sd) reads / sec speedup
nanofilt 01.57 (0.07) 33.48 (0.35) 2,986 01.00 x
filtlong 274.2 (0.04) 16.45 (0.09) 6,079 02.04 x
nanoq 03.68 (0.06) 05.77 (0.04) 17,331 05.80 x
nanoq-fast 03.45 (0.07) 03.20 (0.02) 31,250 10.47 x

Dependencies

Nanoq uses needletail for read operations and niffler for output compression.

Etymology

Avoided name collision with nanoqc and dropped the c to arrive at nanoq [nanɔq] which coincidentally means 'polar bear' in Native American (Eskimo-Aleut, Greenlandic). If you find nanoq useful for your work consider a small donation to the Polar Bear Fund, RAVEN or Inuit Tapiriit Kanatami

Contributions

We welcome any and all suggestions or pull requests. Please feel free to open an issue in the repository on GitHub.

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Minimal but speedy quality control for nanopore reads in Rust 🐻

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