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Manual

Giuseppe Narzisi

scalpel - micro-assembly variant detection tool

CONTENTS
  • SYNOPSIS
  • DESCRIPTION
  • PROTOCOL BUNDLE
  • COMMANDS AND OPTIONS
  • VCF OUTPUT FORMAT
  • ANNOVAR OUTPUT FORMAT
  • AUTHORS
  • LICENSE
  • CITATION
SYNOPSIS

DISCOVERY:

scalpel-discovery --single --bam <BAM file> --bed <BED file> --ref <FASTA file> [OPTIONS]

scalpel-discovery --denovo --dad <BAM file> --mom <BAM file> --aff <BAM file> --sib <BAM file> --bed <BED file> --ref <FASTA file> [OPTIONS]

scalpel-discovery --somatic --normal <BAM file> --tumor <BAM file> --bed <BED file> --ref <FASTA file> [OPTIONS]

EXPORT:

scalpel-export --single --db <DB file> --bed <BED file> --ref <FASTA file> [OPTIONS]

scalpel-export --denovo --db <DB file> --bed <BED file> --ref <FASTA file> [OPTIONS]

scalpel-export --somatic --db <DB file> --bed <BED file> --ref <FASTA file> [OPTIONS]
DESCRIPTION

Scalpel is a software package for detection of indels (insertions and deletions) mutations for
next-generation sequencing data (e.g., Illumina). It supports three modes of operation: single,
denovo, and somatic. In single mode scalpel detects indels in one single dataset (e.g., one individual exome).
In denovo mode scalpel detects de novo indels in one family of four individuals (mom, dad, aff, sib).
In somatic mode scalpel detects somatic indels in a tumor/sample pair given in input.

For all the modes of operation, scalpel requires that the raw reads have been previously aligned with
BWA using default parameters. See BWA description (at http://bio-bwa.sourceforge.net/bwa.shtml) for more info.

PROTOCOL BUNDLE

A protocol bundle (protocol_bundle-0.5.3.tar.gz) is provided with the software package. The bundle contains a complete step-by-step procedure, which can be executed using the master script "run_protocol_0.53.sh", to perform variant calling (de novo and inherited indels) on a HapMap family composed of 4 individuals. Full details about the analysis executed with the bundle is available in the accompanying manuscript:

Fang H, Grabowska EA, Arora K, Vacic V, Zody M, Iossifov I, O’Rawe JA, Y Wu, Jimenez-Barron LT, Rosenbaum J, Ronemus M, Lee Y, Wang Z, Dikoglu E, Jobanputra V, Lyon GJ, Wigler M, Schatz MC, Narzisi G, "Indel variant analysis of short-read sequencing data with Scalpel". Preprint in bioRxiv (2016) doi: http://dx.doi.org/10.1101/028050

COMMANDS AND OPTIONS
single -- variant calling on single sample

usage: scalpel-discovery --single --bam <BAM file=""> --bed <BED file=""> --ref <FASTA file=""> [OPTIONS]

Detect indels in one single dataset (e.g., one individual).

OPTIONS:

--help             : this (help) message
--verbose          : verbose mode

Required:

--bam <BAM file>   : BAM file with the reference-aligned reads
--bed <BED file>   : file with list of regions (BED format) in sorted order or single region in format chr:start-end (example: 1:31656613-31656883)
--ref <FASTA file> : reference genome in FASTA format (same one that was used to create the BAM file)

Optional:

--kmer <int>       : k-mer size [default 25]
--covthr <int>     : threshold used to select source and sink [default 5]
--lowcov <int>     : threshold used to remove low-coverage nodes [default 2]
--covratio <float> : minimum coverage ratio for sequencing errors (default: 0.01)
--radius <int>     : left and right extension (in base-pairs) [default 100]
--window <int>     : window-size of the region to assemble (in base-pairs) [default 400]
--maxregcov <int>  : maximum average coverage allowed per region [default 10000]
--step <int>       : delta shift for the sliding window (in base-pairs) [default 100]
--mapscore <int>   : minimum mapping quality for selecting reads to assemble [default 1]
--pathlimit <int>  : limit number of sequence paths to [default 1000000]
--mismatches <int> : max number of mismatches in near-perfect repeat detection [default 3]
--dir <directory>  : output directory [default ./outdir]
--numprocs <int>   : number of parallel jobs (1 for no parallelization) [default 1]
--sample <string>  : only process reads/fragments in sample [default ALL]
--coords <file>    : file with list of selected locations to examine [default null]

Output:

--format           : export mutations in selected format (annovar | vcf) [default vcf]
--intarget         : export mutations only inside the target regions from the BED file
--logs             : keep log files

Note 1: the list of detected indels is saved in file: OUTDIR/variants.*.indel.*
where OUTDIR is the output directory selected with option "--dir" [default ./outdir]

Note 2: use the export tool (scalpel-export) to export mutations using different filtering criteria
denovo -- call de novo variants in one family of four individuals (mom, dad, aff, sib)

usage: scalpel-discovery --denovo --dad <BAM file=""> --mom <BAM file=""> --aff <BAM file=""> --sib <BAM file=""> --bed <BED file=""> --ref <FASTA file=""> [OPTIONS]

Detect de novo indels in a family of four individuals (mom, dad, aff, sib).

OPTIONS:

--help             : this (help) message
--verbose          : verbose mode

Required:

--dad <BAM file>   : father BAM file
--mom <BAM file>   : mother BAM file
--aff <BAM file>   : affected child BAM file
--sib <BAM file>   : sibling BAM file
--bed <BED file>   : file with list of regions (BED format) in sorted order or single region in format chr:start-end (example: 1:31656613-31656883)
--ref <FASTA file> : reference genome in FASTA format (same one that was used to create the BAM file)

Optional:

--kmer <int>       : k-mer size [default 25]
--covthr <int>     : threshold used to select source and sink [default 5]
--lowcov <int>     : threshold used to remove low-coverage nodes [default 2]
--covratio <float> : minimum coverage ratio for sequencing errors (default: 0.01)
--radius <int>     : left and right extension (in base-pairs) [default 100]
--window <int>     : window-size of the region to assemble (in base-pairs) [default 400]
--maxregcov <int>  : maximum average coverage allowed per region [default 10000]
--step <int>       : delta shift for the sliding window (in base-pairs) [default 100]
--mapscore <int>   : minimum mapping quality for selecting reads to assemble [default 1]
--pathlimit <int>  : limit number of sequence paths to [default 1000000]
--mismatches <int> : max number of mismatches in near-perfect repeat detection [default 3]
--dir <directory>  : output directory [default ./outdir]
--numprocs <int>   : number of parallel jobs (1 for no parallelization) [default 1]
--coords <file>    : file with list of selected coordinates to examine [default null]
--two-pass         : perform second pass of analysis to confirm candidate calls

Output:

--format           : export mutations in selected format (annovar | vcf) [default vcf]
--intarget         : export mutations only inside the target regions from the BED file
--logs             : keep log files

Note 1: the list of de novo indels is saved in file: OUTDIR/denovos..indel.
where OUTDIR is the output directory selected with option "--dir" [default ./outdir]

Note 2: use the export tool (scalpel-export) to export mutations using different filtering criteria

somatic - detects somatic indels in a tumor/normal pair

usage: scalpel-discovery --somatic --normal <BAM file=""> --tumor <BAM file=""> --bed <BED file=""> --ref <FASTA file=""> [OPTIONS]

Detect somatic indels in a tumor/normal pair

OPTIONS:

--help                : this (help) message
--verbose             : verbose mode

Required:

--normal <BAM file>   : normal BAM file
--tumor  <BAM file>   : tumor BAM file
--bed    <BED file>   : file with list of regions (BED format) in sorted order or single region in format chr:start-end (example: 1:31656613-31656883)
--ref    <FASTA file> : reference genome in FASTA format (same one that was used to create the BAM file)

Optional:

--kmer <int>          : k-mer size [default 25]
--covthr <int>        : threshold used to select source and sink [default 5]
--lowcov <int>        : threshold used to remove low-coverage nodes [default 2]
--covratio <float>    : minimum coverage ratio for sequencing errors (default: 0.01)
--radius <int>        : left and right extension (in base-pairs) [default 100]
--window <int>        : window-size of the region to assemble (in base-pairs) [default 400]
--maxregcov <int>     : maximum average coverage allowed per region [default 10000]
--step <int>          : delta shift for the sliding window (in base-pairs) [default 100]
--mapscore <int>      : minimum mapping quality for selecting reads to assemble [default 1]
--pathlimit <int>     : limit number of sequence paths to [default 1000000]
--mismatches <int>    : max number of mismatches in near-perfect repeat detection [default 3]
--dir <directory>     : output directory [default ./outdir]
--numprocs <int>      : number of parallel jobs (1 for no parallelization) [default 1]
--coords <file>       : file with list of selected coordinates to examine [default null]
--two-pass            : perform second pass of analysis to confirm candidate calls

Output:

--format              : export mutations in selected format (annovar | vcf) [default vcf]
--intarget            : export mutations only inside the target regions from the BED file
--logs                : keep log files

Note 1: the list of somatic indels is saved in file: OUTDIR/somatic.indel.*
where OUTDIR is the output directory selected with option "--dir" [default ./outdir].

Note 2: use the export tool (scalpel-export) to export mutations using different filtering criteria

EXPORT TOOL

Export mutations from database in single, somatic or denovo mode

export -- single mode

usage: scalpel-export --single --db <file> --bed <file> --ref <file> [OPTIONS]

OPTIONS:

--help             : this (help) message
--verbose          : verbose mode

Required:

--db <file>        : Database of mutations
--bed <file>       : file with list of regions (BED format) in sorted order or single region in format chr:start-end (example: 1:31656613-31656883)
--ref <FASTA file> : reference genome in FASTA format (same one that was used to create the BAM file)

Optional:

--output-format <text>   : output format for variants (annovar | vcf) [default vcf]
--variant-type <text>    : mutation type (snp, del, ins, indel, all: everything) [default indel]
--min-ins-size <int>     : minimum size of an insertion [default 1]
--max-ins-size <int>     : maximum size of an insertion [default 1000000000]
--min-del-size <int>     : minimum size of a deletion [default 1]
--max-del-size <int>     : maximum size of a deletion [default 1000000000]
--min-alt-count <int>    : minimum alternative count [default 5]
--max-alt-count <int>    : maximum alternative count [default 1000000]
--min-coverage <int>     : minimum coverage [default 5]
--max-coverage <int>     : maximum coverage [default 1000000]
--min-chi2-score <float> : minimum chi-square score [default 0]
--max-chi2-score <float> : maximum chi-square score [default 20]
--min-vaf <float>        : minimum variant allele frequency (AlleleCov/TotCov) [default 0.05]
--intarget               : export mutations only inside the target regions from the BED file

Supported output formats:
1. annovar
2. vcf

NOTE: The database.db file can be found in the output directory for the single operation
mode or in the correspective subdirectories ("main" and "twopass' for denovo and soamtic modes).
export -- denovo mode

usage: scalpel-export --denovo --db <file> --bed <file> --ref <file> [OPTIONS]

OPTIONS:

--help             : this (help) message
--verbose          : verbose mode

Required:

--db <file>        : Database of mutations
--bed <file>       : file with list of regions (BED format) in sorted order or single region in format chr:start-end (example: 1:31656613-31656883)
--ref <FASTA file> : reference genome in FASTA format (same one that was used to create the BAM file)

Optional:

--output-format <text>           : output format for variants (annovar | vcf) [default vcf]
--variant-type <text>            : mutation type (snp, del, ins, indel, all: everything) [default indel]
--min-ins-size <int>             : minimum size of an insertion [default 1]
--max-ins-size <int>             : maximum size of an insertion [default 1000000000]
--min-del-size <int>             : minimum size of a deletion [default 1]
--max-del-size <int>             : maximum size of a deletion [default 1000000000]
--min-alt-count-affected <int>   : minimum alternative count in the affected sample [default 5]
--max-alt-count-unaffected <int> : maximum alternative count in the unaffected samples [default 0]
--min-vaf-affected <float>       : minimum variant allele frequency (AlleleCov/TotCov) in the affected sample [default 0.05]
--max-vaf-unaffected <float>     : maximum variant allele frequency (AlleleCov/TotCov) in the unaffected samples [default 0]
--min-coverage-affected <int>    : minimum coverage in the affected sample [default 5]
--max-coverage-affected <int>    : maximum coverage in the affected sample [default 1000000000]
--min-coverage-unaffected <int>  : minimum coverage in the unaffected samples [default 10]
--max-coverage-unaffected <int>  : maximum coverage in the unaffected samples [default 1000000000]
--min-chi2-score <float>         : minimum chi-square score [default 0]
--max-chi2-score <float>         : maximum chi-square score [default 20]
--intarget                       : export mutations only inside the target regions from the BED file

Supported output formats:

1. annovar
2. vcf

NOTE: The database.db file can be found in the output directory for the single operation
mode or in the correspective subdirectories ("main" and "twopass' for denovo and soamtic modes).
export -- somatic mode

usage: scalpel-export --somatic --db <file> --bed <file> --ref <file> [OPTIONS]

OPTIONS:

--help             : this (help) message
--verbose          : verbose mode

Required:

--db <file>        : Database of mutations
--bed <file>       : file with list of regions (BED format) in sorted order or single region in format chr:start-end (example: 1:31656613-31656883)
--ref <FASTA file> : reference genome in FASTA format (same one that was used to create the BAM file)

Optional:

--output-format <text>       : output format for variants (annovar | vcf) [default vcf]
--variant-type <text>        : mutation type (snp, del, ins, indel, all: everything) [default indel]
--min-ins-size <int>         : minimum size of an insertion [default 1]
--max-ins-size <int>         : maximum size of an insertion [default 1000000000]
--min-del-size <int>         : minimum size of a deletion [default 1]
--max-del-size <int>         : maximum size of a deletion [default 1000000000]
--min-alt-count-tumor <int>  : minimum alternative count in the tumor [default 4]
--max-alt-count-normal <int> : maximum alternative count in the normal [0]
--min-vaf-tumor <float>      : minimum variant allele frequency (AlleleCov/TotCov) in the tumor [default 0.05]
--max-vaf-normal <float>     : maximum variant allele frequency (AlleleCov/TotCov) in the normal, maximum value allowed 0.1 [default 0]
--min-coverage-tumor <int>   : minimum coverage in the tumor [default 4]
--max-coverage-tumor <int>   : maximum coverage in the tumor [default 1000000000]
--min-coverage-normal <int>  : minimum coverage in the normal [default 10]
--max-coverage-normal <int>  : maximum coverage in the normal [default 1000000000]
--min-phred-fisher <float>   : minimum fisher exact test score [default 10]
--intarget                   : export mutations only inside the target regions from the BED file

Supported output formats:

1. annovar
2. vcf

NOTE: The database.db file can be found in the output directory for the single operation
mode or in the correspective subdirectories ("main" and "twopass' for denovo and soamtic modes).

By default scalpel exports the list of detected indels in a file named variants..indel. in the
selected output directory according to default filtering parameters.
However it is recommended to explore different filtering criteria using the export tool.
Three important parameters are:

  1. mininum altertnative count:
    • Change this number according to the type of study (germline, somatic, denovo) and the expected coverage.
    • Smaller values will give more sensitivity but increase the number of false-positive calls.
  2. minimum variant allele frequency VAF (altCoverage/totalCoverage)
    • Similarly to the minimum alternative count parameter, smaller values will increase sensitivity but reduce specificity.
  3. maximum chi-squared score:
    • Chi square test statistic computed using the reference and alternative coverage for the mutation.
    • Larger values will give more sensitivity but produce a large number of false-positives.
    • For germline and denovo discovery we recommend using chi-square score ≤ 20 to select high confidence indels.
  4. minimum fisher exact test score:
    • Fisher exact test statistic computed using the reference and alternative counts in tumor and normal samples.
    • Goal is to test the independence between the allele balances in the tumor and the normal.
    • We recommend using a fisher score > 10 to select high confidence somatic indels.
VCF OUTPUT FORMAT

The list of candidate mutations is exported according to VCF format v4.1.
Following the VCF best practices, high quality calls are marked as "PASS" in the FILTER column.
Multi-sample VCFs are generated for somatic and denovo analysis
Several filters are applyed to variants according to the input paramteres.
If a variant is not labelled as "PASS", the FILTER info field will provide the list of applyed filters:

single mode filters:

##FILTER=<ID=MS,Description="Microsatellite mutation="" (format:="" #LEN#MOTIF)"="">
##FILTER=<ID=LowVaf,Description="low variant="" allele="" frequency="" (<0.05)"="">
##FILTER=<ID=LowAltCnt,Description="low alternative="" allele="" count="" (<5)"="">
##FILTER=<ID=HighAltCnt,Description="high alternative="" allele="" count="" (="">1000000)">
##FILTER=<ID=LowChi2score,Description="low chi-squared="" score="" (<0)"="">
##FILTER=<ID=HighChi2score,Description="high chi-squared="" score="" (="">20)">

denovo mode filters:

##FILTER=<ID=MS,Description="Microsatellite mutation="" (format:="" #LEN#MOTIF)"="">
##FILTER=<ID=LowCovAff,Description="low coverage="" in="" affected="" sample="" (<5)"="">
##FILTER=<ID=LowCovUnaff,Description="low coverage="" in="" unaffected="" samples="" (<10)"="">
##FILTER=<ID=HighCovAff,Description="high coverage="" in="" affected="" sample="" (="">1000000000)">
##FILTER=<ID=HighCovUnaff,Description="high coverage="" in="" unaffected="" samples="" (="">1000000000)">
##FILTER=<ID=LowVafAff,Description="low variant="" allele="" frequency="" for="" affected="" sample="" (<0.05)"="">
##FILTER=<ID=HighVafUnaff,Description="high variant="" allele="" frequency="" for="" unaffected="" sample="" (="">0)">
##FILTER=<ID=LowAltCntAff,Description="low alternative="" allele="" count="" for="" affected="" sample="" (<5)"="">
##FILTER=<ID=HighAltCntUnaff,Description="high alternative="" allele="" count="" for="" unaffected="" sample="" (="">0)">
##FILTER=<ID=LowChi2score,Description="low chi-squared="" score="" (<0)"="">
##FILTER=<ID=HighChi2score,Description="high chi-squared="" score="" (="">1)">

somatic moded filters:

##FILTER=<ID=MS,Description="Microsatellite mutation="" (format:="" #LEN#MOTIF)"="">
##FILTER=<ID=LowCovNormal,Description="low coverage="" in="" the="" normal="" (<10)"="">
##FILTER=<ID=HighCovNormal,Description="high coverage="" in="" the="" normal="" (="">1000000000)">
##FILTER=<ID=LowCovTumor,Description="low coverage="" in="" the="" tumor="" (<4)"="">
##FILTER=<ID=HighCovTumor,Description="high coverage="" in="" the="" tumor="" (="">1000000000)">
##FILTER=<ID=LowVafTumor,Description="low variant="" allele="" frequency="" for="" tumor="" (<0.05)"="">
##FILTER=<ID=HighVafNormal,Description="high variant="" allele="" frequency="" for="" normal="" (="">0)">
##FILTER=<ID=LowAltCntTumor,Description="low alternative="" allele="" count="" for="" tumor="" (<4)"="">
##FILTER=<ID=HighAltCntNormal,Description="high alternative="" allele="" count="" for="" normal="" (="">0)">
##FILTER=<ID=LowFisherScore,Description="low fisher="" exact="" test="" score="" for="" tumor-normal="" (<10)"="">

The INFO fields contain the following (somehow redundant) information:

- avgKCov     : average k-mer coverage for the mutation
- minKCov     : minimum k-mer coverage for the mutation (can be different from avgKCov for insertions)
- zygosity    : homozygous or heterozygous
- altKCov     : coverage of alternative alleles at locus (>0 for heterozygous mutation)
- covRatio    : coverage ratio computed as minKCov/(minKCov+altKCov)
- chi2score   : chi-squared statistic (based on coverage), it is recommended to use chi-square score ≤ 20 for high confidence indels.
- inheritance : if mutation is found in parents (for --denovo mode) or if found in normal (for --somatic mode)
- bestState   : state of the mutations as described below (only for --denovo and --somatic mode)  
- covState    : coverage state of the mutation as described below (only for --denovo and --somatic mode)

The format of "bestState" for --denovo mode is:

"momR dadR affR sibR/momA dadA affA sibA"

where (for example) momR stands for the number of copies of the reference allele
in the mother’s genotype and affA stands for the number of copies of the alternative
allele in the genotype of the affected child.

Similarly, the format of "bestState" for --somatic mode is:

"normalR tumorR/normalA tumorA"

where (for example) normalR stands for the number of copies of the reference allele
in the normal’s genotype and tumorA stands for the number of copies of the alternative
allele in the genotype of the tumor.

The format of "covState" for --denovo mode is:

"momRcov dadRcov affRcov sibRcov/momAcov dadAcov affAcov sibAcov"

where (for example) "momRcov" stands for the k-mer coverage of the reference allele
in the mother’s genotype and "affAcov" stands for the k-mer coverage of the alternative
allele in the genotype of the affected child.

Similarly, the format of "covState" for --somatic mode is:

"normalRcov tumorRcov/normalAcov tumorAcov"

where (for example) "normalRcov" stands for the k-mer coverage of the reference allele
in the normal’s genotype and "tumorAcov" stands for the k-mer coverage of the alternative
allele in the genotype of the tumor.

ANNOVAR OUTPUT FORMAT (obsolete)

The list of candidate mutations is exported according to the following format.
Columns 1 to 5 describe the mutation according to the "annovar" format (http://www.openbioinformatics.org/annovar/).

The columns description are:

- chr         : chromosome
- start       : start position
- end         : end position
- ref         : reference nucleotides
- obs         : observed nucleotides
- id          : id of the sample where the mutation is (only for --denovo and --somatic mode)
- size        : size (in nt) of the mutation.
- type        : deletion or insertion
- avgKCov     : average k-mer coverage for the mutation
- minKCov     : minimum k-mer coverage for the mutation (can be different from avgKCov for insertions)
- zygosity    : homozygous or heterozygous
- altKCov     : coverage of alternative alleles at locus (>0 for heterozygous mutation)
- covRatio    : coverage ratio computed as minKCov/(minKCov+altKCov)
- chi2score   : chi-squared statistic (based on coverage), it is recommended to use chi-square score ≤ 20 for high confidence indels.
- inheritance : if mutation is found in parents (for --denovo mode) or if found in normal (for --somatic mode)
- bestState   : state of the mutations as described below (only for --denovo and --somatic mode)  
- covState    : coverage state of the mutation as described below (only for --denovo and --somatic mode)
AUTHORS
Giuseppe Narzisi and Michael C. Schatz, Cold Spring Harbor Laboratory (CSHL).
LICENSE
The Scalpel package is distributed under the MIT License.
CITATION
Narzisi G., O'Rawe J.A., Iossifov I., Fang H., Lee Y., Wang Z., Wu Y., Lyon G.J., Wigler M., Schatz M.C. 
Accurate de novo and transmitted indel detection in exome-capture data using microassembly. 
Nature Methods 11, 1033–1036 (2014) (DOI: 10.1038/nmeth.3069)