rustynuc

Tool to calculate the likelihood of 8-oxoG damage based on alignment characteristics.

Install

Conda

To install with conda:

conda install -c bioconda rustynuc

Binary

Precompiled binaries are provided below:

TAR	TAR
ZIP	ZIP

Cargo

If you have cargo installed or have installed RUSTUP, you can install directly from:

• Crates.io

cargo install rustynuc

• Github

cargo install --git https://github.com/bjohnnyd/rustynuc

Build

To compile from source rustup is required and can be obtained HERE. After installing rustup download the release archive file and build:

git clone https://github.com/bjohnnyd/rustynuc.git && cd rustynuc && cargo build --release

All releases and associated binaries and archives are accessible under Releases.

Usage

./rustynuc -h

rustynuc 0.3.0

USAGE:
    rustynuc [FLAGS] [OPTIONS] <bam>

FLAGS:
    -a, --all                Whether to process and print information for every position in the BAM file
    -h, --help               Prints help information
        --no-overlapping     Do not count overlapping mates when calculating total depth
    -n, --no-qval            Skip calculating qvalue
    -p, --pseudocount        Whether to use pseudocounts (increments all counts by 1) when calculating statistics
        --skip-fishers       Skip applying Fisher's Exact Filter on VCF
    -V, --version            Prints version information
    -v, --verbosity          Determines verbosity of the processing, can be specified multiple times -vvv
    -w, --with-track-line    Include track line (for correct visualization with IGV)

OPTIONS:
        --af-both-pass <af-both-pass>        AF on both the ff and fr at which point a call in the VCF will excluded
                                             from the OxoAF filter [default: 0.1]
        --af-either-pass <af-either-pass>    AF above this cutoff in EITHER read orientation will be excluded from OxoAF
                                             filter [default: 0.25]
        --alpha <alpha>                      FDR threshold [default: 0.2]
    -b, --bcf <bcf>                          BCF/VCF for variants called on the supplied alignment file
        --bed <bed>                          A BED file to restrict analysis to specific regions
        --fishers-sig <fishers-sig>          Significance threshold for Fisher's test [default: 0.05]
        --max-depth <max-depth>              Maximum pileup depth to use [default: 1000]
    -m, --min-reads <min-reads>              Minimum number of aligned reads in ff or fr orientation for a position to
                                             be considered [default: 4]
    -q, --quality <quality>                  Minimum base quality to consider [default: 20]
    -r, --reference <reference>              Optional reference which will be used to determine sequence context and
                                             type of change
    -t, --threads <threads>                  Number of threads [default: 4]

ARGS:
    <bam>    Alignments to investigate for possible 8-oxoG damage

Output

The default output (if no --bcf/-b is provided) is a BED file with the following info:

1. Chromosome
2. Start
3. End
4. Name (format is `<chromosome>_<start>_<end>` or if reference is provided `<chromosome>_<base>_<start>_<end>`
5. -log10 of p-value (p-value is the smallest of the A/C and G/T )
6. Strand
7. Depth
8. Adenine FF:FR counts
9. Cytosine FF:FR counts
10. Guanine FF:FR counts
11. Thymine FF:FR counts
12. A/C two-sided p-value Fisher's Exact Test
13. G/T two-sided p-value Fisher's Exact Test
(14). Sequnce Context (if reference provided)
14/15. adj. pvalue
15/16. Significant at set FDR value (1 if yes, 0 if not)

To get only positions with p-value below 0.05:

rustynuc -r tests/input/ref.fa.gz tests/alignments/oxog.bam | awk '$12 < 0.05 || $13 < 0.05'  | gzip > sig.bed.gz

If a VCF/BCF is provided the output will be in VCF format. Multiple summaries are provided in the VCF file:

AF_FF_FR can be used to filter based on AF on the FF or FR orientations.

For each alternate allele, there are two AF provided so for example to filter the first alternate positions AF_FF_FR[0] and AF_FF_FR[1] can be used. The command below will filter using the AF on FF/FR and also FILTER=="PASS" ensures only position with p-val < 0.05 are returned.

FILTERCMD='TYPE =="snp" && AF > 0.04 && FILTER=="PASS" && (FF_FR_AF=="." || (FF_FR_AF[0] >= 0.04 && FF_FR_AF[1] >= 0.04))'
rustynuc --pseudocounts -r tests/input/ref.fa.gz --b tests/input/oxog.vcf.gz tests/alignments/oxog.bam | bcftools filter -Oz -i "$FILTERCMD" > nonoxog.vcf.gz

Authors

• Johnny Debebe

License

The MIT License (MIT). Please see License File for more information.

Notes

Currently will only process non-MNP calls so it is recommended to normalize and convert to allelic primitives all variants prior to using the tool.

Additional Notes

• DEPTH is the key determinant of power in discerning 8-oxoG
• in cases where depth is not high the AF_FF_FR alternate frequency filter is a better option
• fisher's exact is affected by 0 counts so pseudocounts can be used
• FDR will be heavily dependent on %GC of the genome, size of the genome, whether a reference was provided, a VCF is provided or the test was restricted to specific regions.

Crates to Credit

Implemented using the rust-htslib and niffler crates.

Citing

If used in published research, a citation is appreciated:

Debebe, Bisrat J: Quick analysis of pileups for likely 8-oxoG locations. (2020). doi:10.5281/zenodo.4157557