# FastaAlternateReferenceMaker

Generates an alternative reference sequence over the specified interval.

## Overview

Given variant tracks, it replaces the reference bases at variation sites with the bases supplied by the ROD(s). Additionally, allows for one or more "snpmask" VCFs to set overlapping bases to 'N'. The output format can be partially controlled using the provided command-line arguments. Specify intervals with the usual -L argument to output only the reference bases within your intervals. Overlapping intervals are automatically merged; reference bases for each disjoint interval will be output as a separate fasta sequence (named numerically in order). Several important notes: 1) if there are multiple variants that start at a site, it chooses one of them randomly. 2) when there are overlapping indels (but with different start positions) only the first will be chosen. 3) this tool works only for SNPs and for simple indels (but not for things like complex substitutions). Reference bases for each interval will be output as a separate fasta sequence (named numerically in order).

### Input

The reference, requested intervals, and any number of variant rod files.

### Output

A fasta file representing the requested intervals.

### Examples

 java -Xmx2g -jar GenomeAnalysisTK.jar \
-R ref.fasta \
-T FastaAlternateReferenceMaker \
-o output.fasta \
-L input.intervals \
--variant input.vcf \


These Read Filters are automatically applied to the data by the Engine before processing by FastaAlternateReferenceMaker.

### Downsampling settings

This tool applies the following downsampling settings by default.

• Mode: BY_SAMPLE
• To coverage: 1,000

### Window size

This tool uses a sliding window on the reference.

• Window start: -1 bp before the locus
• Window stop: 50 bp after the locus

## Command-line Arguments

### Inherited arguments

The arguments described in the entries below can be supplied to this tool to modify its behavior. For example, the -L argument directs the GATK engine restricts processing to specific genomic intervals (this is an Engine capability and is therefore available to all GATK walkers).

### FastaAlternateReferenceMaker specific arguments

This table summarizes the command-line arguments that are specific to this tool. For more details on each argument, see the list further down below the table or click on an argument name to jump directly to that entry in the list.

Argument name(s) Default value Summary
Required Inputs
--variant
-V
NA Input VCF file
Optional Inputs
Optional Outputs
--out
-o
NA An output file created by the walker. Will overwrite contents if file exists
Optional Parameters
--lineWidth
-lw
60 Maximum length of sequence to write per line
--use_IUPAC_sample
-IUPAC
NA If specified, heterozygous SNP sites will be output using IUPAC ambiguity codes given the genotypes for this sample
Optional Flags
--rawOnelineSeq
-raw
NA Print sequences with no FASTA header lines, one line per interval (i.e. lineWidth = infinity)

### Argument details

Arguments in this list are specific to this tool. Keep in mind that other arguments are available that are shared with other tools (e.g. command-line GATK arguments); see Inherited arguments above.

### --lineWidth / -lw

Maximum length of sequence to write per line

int  [ [ -∞  ∞ ] ]

### --out / -o

An output file created by the walker. Will overwrite contents if file exists

PrintStream

### --rawOnelineSeq / -raw

Print sequences with no FASTA header lines, one line per interval (i.e. lineWidth = infinity)
Please note that when using this argument adjacent intervals will automatically be merged.

boolean

Snps from this file are used as a mask (inserting N's in the sequence) when constructing the alternate reference (regardless of whether they overlap a variant site).

--snpmask binds reference ordered data. This argument supports ROD files of the following types: BCF2, VCF, VCF3

RodBinding[VariantContext]

### --use_IUPAC_sample / -IUPAC

If specified, heterozygous SNP sites will be output using IUPAC ambiguity codes given the genotypes for this sample
This option will generate an error if the specified sample does not exist in the VCF. Non-diploid (or non-called) genotypes are ignored.

String

### --variant / -V

Input VCF file
Variants from this VCF file are used by this tool as input. The file must at least contain the standard VCF header lines, but can be empty (i.e., no variants are contained in the file).

--variant binds reference ordered data. This argument supports ROD files of the following types: BCF2, VCF, VCF3

R RodBinding[VariantContext]