2. Usage Instructions
- Input and output parameters
- How to run CHERIPIC - command line
- How to run CHERIPIC - web app
- Details about parameters
- Output
- Test Data
Running cheripic without any input at command line interface shows following help options
Cheripic v1.2.6
Authors: Shyam Rallapalli Martin Page and Dan MacLean
Description: Candidate mutation and closely linked marker selection for non reference genomes
Uses bulk segregant data from non-reference sequence genomes
Inputs:
1. Needs a reference fasta file of asssembly use for variant analysis
2. Pileup/Bam files for mutant (phenotype of interest) bulks and background (wildtype phenotype) bulks
3. If providing bam files, you have to include vcf files for the respective bulks
4. If polyploid species, include pileup/bam files from one or both parents
USAGE:
cheripic <options>
OPTIONS:
-f, --assembly=<s> Assembly file in FASTA format
-F, --input-format=<s> bulk and parent alignment file format types - set either pileup or bam or vcf (default: pileup)
-a, --mut-bulk=<s> Pileup or sorted BAM file alignments from mutant/trait of interest bulk 1
--mut-bulk-vcf=<s> vcf file for variants from mutant/trait of interest bulk 1 (default: )
-b, --bg-bulk=<s> Pileup or sorted BAM file alignments from background/wildtype bulk 2
--bg-bulk-vcf=<s> vcf file for variants from background/wildtype bulk 2 (default: )
--output=<s> custom name tag to include in the output file name (default: cheripic_results)
--loglevel=<s> Choose any one of "info / warn / debug" level for logs generated (default: debug)
--hmes-adjust=<f> factor added to snp count of each contig to adjust for hme score calculations (default: 0.5)
--htlow=<f> lower level for categorizing heterozygosity (default: 0.2)
--hthigh=<f> high level for categorizing heterozygosity (default: 0.9)
--mindepth=<i> minimum read depth at a position to consider for variant calls (default: 6)
--max-d-multiple=<i> multiplication factor for average coverage to calculate maximum read coverage
if set zero no calculation will be made from bam file.
setting this value will override user set max depth (Default: 5)
--maxdepth=<i> maximum read depth at a position to consider for variant calls
if set to zero no user max depth will be used (default: 0)
--min-non-ref-count=<i> minimum read depth supporting non reference base at each position (default: 3)
--min-indel-count-support=<i> minimum read depth supporting an indel at each position (default: 3)
--ambiguous-ref-bases=<s> including variant at completely ambiguous bases in the reference (default: false)
-q, --mapping-quality=<i> minimum mapping quality of read covering the position (default: 20)
-Q, --base-quality=<i> minimum base quality of bases covering the position (default: 15)
--noise=<f> praportion of reads for a variant to conisder as noise (default: 0.1)
--cross-type=<s> type of cross used to generated mapping population - back or out (default: back)
--use-all-contigs=<s> option to select all contigs or only contigs containing variants for analysis (default: false)
--include-low-hmes=<s> option to include or discard variants from contigs with
low hme-score or bfr score to list in the final output (default: false)
--polyploidy=<s> Set if the data input is from polyploids (default: false)
-p, --mut-parent=<s> Pileup or sorted BAM file alignments from mutant/trait of interest parent (default: )
-r, --bg-parent=<s> Pileup or sorted BAM file alignments from background/wildtype parent (default: )
-R, --repeats-file=<s> repeat masker output file for the assembly (default: )
--bfr-adjust=<f> factor added to hemi snp frequency of each parent to adjust for bfr calculations (default: 0.05)
--sel-seq-len=<i> sequence length to print from either side of selected variants (default: 50)
--examples shows some example commands with explanation
cheripic -f assembly.fa -a mutbulk.pileup -b bgbulk.pileup --output=cheripic_output
or
cheripic --assembly assembly.fa --mut-bulk mutbulk.pileup --bg-bulk bgbulk.pileup --output cheripic_results
Use of vcf file as input
cheripic --assembly assembly.fa --input-format vcf --mut-bulk mutbulk.vcf --bg-bulk bgbulk.vcf --output cheripic_results
Use of vcf file as input
cheripic --assembly assembly.fa --input-format vcf --mut-bulk mutbulk.vcf --bg-bulk bgbulk.vcf --output cheripic_results
cheripic --assembly assembly.fa --mut-bulk mutbulk.pileup --bg-bulk bgbulk.pileup
--mut-parent mutparent.pileup --bg-parent bgparent.pileup --polyploidy true --output cheripic_results
CHERIPIC web app is available at http://cheripic.tsl.ac.uk/
-f, --assembly
Assembly file in FASTA format
-F, --input-format
bulk and parent alignment file format types - set either pileup or vcf or bam (default: pileup)
-a, --mut-bulk
Pileup or sorted BAM file alignments from mutant/trait of interest bulk 1
-b, --bg-bulk
Pileup or sorted BAM file alignments from background/wild type bulk 2
--mut-bulk-vcf
vcf file for variants from mutant/trait of interest bulk 1. This is needed when bulk input-format is bam
--bg-bulk-vcf
vcf file for variants from background/wild type bulk 2. This is needed when bulk input-format is bam
--output
custom name tag to include to the output file name (default: cheripic_results)
--loglevel
Choose any one of "info / warn / debug" level for logs generated (default: debug)
--hmes-adjust
factor added to variant count of each contig to adjust for HMES calculations, especially deal with divisions with zeros (default: 0.5)
--htlow
Lower limit of allele frequency to categorise a variant as heterozygous (default: 0.2)
--hthigh
Upper limit of allele frequency to categorise a variant as heterozygous (default: 0.9)
--mindepth
Minimum read depth at a position to be consider as variant for downstream analysis (default: 6)
--max-d-multiple
Multiplication factor for average coverage to calculate maximum read coverage. If set zero no calculation will be made from bam file. Setting this value will override user set max depth (Default: 5)
--maxdepth
Maximum read depth at a position to consider for variant calls. If set to zero no user max depth will be used (default: 0)
--min-non-ref-count
minimum read depth supporting non reference base at each position (default: 3)
--min-indel-count-support
minimum read depth supporting an indel at each position (default: 3)
--ambiguous-ref-bases
including variant at completely ambiguous bases in the reference (default: false)
-q, --mapping-quality
minimum mapping quality of read covering the position (default: 20)
-Q, --base-quality
minimum base quality of bases covering the position (default: 15)
--noise
proportion of reads for a variant to consider as noise (default: 0.1)
--cross-type
type of cross used to generated mapping population - back or out (default: back)
--use-all-contigs
option to select all contigs or only contigs containing variants for analysis (default: false)
--include-low-hmes
option to include or discard variants from contigs with low hme-score or bfr score to list in the final output (default: false)
--polyploidy
Set if the data input is from polyploids (default: false)
-p, --mut-parent
Pileup or sorted BAM file alignments from mutant/trait of interest parent (default: )
-r, --bg-parent
Pileup or sorted BAM file alignments from background/wildtype parent (default: )
-R, --repeats-file
repeat masker output file for the assembly (default: )
--bfr-adjust
factor added to hemi snp frequency of each parent to adjust for bfr calculations (default: 0.05)
--sel-seq-len
sequence length to print from either side of selected variants (default: 50)
--examples
shows some example commands with explanation
Output from CHERIPIC is a tab-delimited file with following 12 columns of information about the variants selected -
- HMES - homozygosity enrichment score of the selected variant
- AlleleFreq - allele frequency at variant position,
- length - length of the contig,
- seq_id - id of the contig,
- position - variant position in the contig,
- ref_base - reference base,
- coverage - read coverage
- bases - read bases
- base_quals - base qualities,
- sequence_left - sequence on the left side of variant,
- Alt_seq - variant allele,
- sequence_right - sequence on the right side of variant.
Left and right sequences are provided to easily design markers and sequence lengths can user adjusted to retrieve enough sequence information. By default 50 bases on either side of variant are provided, this can be edited to retrieve longer sequences.
-
Test data folder has following files
- input_assembly_file.fa
- mutant_bam_file.bam
- mutant_pileup_file.pileup
- mutant_vcf_file.vcf
- wildtype_bam_file.bam
- wildtype_pileup_file.pileup
- wildtype_vcf_file.vcf
-
input_assembly_file.faas name suggests is the assembly file to use -
mutant and wild type bulks files start with
mutant_andwildtype_, respectively, in their file names. -
We have provided pileup, vcf and bam files for mutant bulks and wild type bulks to be able to test using different bulk input options
-
Example commands to use data in this folder
pileup inputs
./cheripic -f test_data/input_assembly_file.fa -a test_data/mutant_pileup_file.pileup -b test_data/wildtype_pileup_file.pileupvcf inputs
./cheripic -F vcf -f test_data/input_assembly_file.fa -a test_data/mutant_vcf_file.vcf -b test_data/wildtype_vcf_file.vcf