QTLtools-ase

NAME

QTLtools ase - Measure ASE from RNA-seq

SYNOPSIS

QTLtools ase --bam [sample.bam|sample.sam|sample.cram] --vcf [in.vcf|in.bcf|in.vcf.gz] --sample sample_name_in_vcf --mapq integer --out output_file_prefix [OPTIONS]

DESCRIPTION

This mode measures allele specific expression (ASE) from RNAseq for transcribed heterozygous SNPs as detailed in <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918453/>. In brief, the reference allele mapping bias is calculated for each of the 12 REF/ALT pairs separately provided that there are at least --sites number of REF/ALT sites that have a minimum --cov-bias number of reads overlapping. For REF/ALT pairs that fail these criteria, reference allele mapping bias is calculated from all sites. Reference allele mapping bias is the number of all reads across many sites that contained the reference allele for a given REF/ALT pair over the total number of reads overlapping. --subsample controls which percentile of the highest covered sites are subsampled, which is necessary so that the reference allele mapping bias is not estimated mostly from very high coverage sites, but from sites covering all the genome. The ASE p-value for each site is then calculated as a two-tailed binomial test checking if the observed number of reference allele reads is significantly different from random, given the total number of reads, and the probability of observing a reference allele, which is the reference allele mapping bias for a certain REF/ALT pair.

The defaults for options should work well for most RNAseq experiments. It is NOT advisable to decrease the --baseq below 10, --cov and --cov-bias below 8, and setting --subsample to 1. It is NOT recommended to use the following options regarding filtering --keep-bans-for-bias, --keep-discordant-for-bias, --filter-duplicates, --ignore-orientation, and --legacy-options. Also refrain from using --auto-flip, but rather create correct VCF/BCF files.

We highly recommended using a BCF file rather than a VCF due to performance benefits, --fasta to provide the genome sequence used, --blacklist to filter low mappability regions, and --gtf to annotate the SNPs. If you are using unfiltered imputed genotypes then consider using --imp-qual and --geno-prob. For trouble shooting purposes you can use --filtered, which will list why certain variants are filtered from the analysis. If you are I/O bound you may try using --group-by for better performance. If your blacklist file contains many overlapping or contiguous regions you can decrease the memory usage with --merge-on-the-fly.

OPTIONS

--vcf, -v [in.vcf|in.bcf|in.vcf.gz]

Genotypes in VCF/BCF format sorted by chromosome and then position. Can contain multiple samples. REQUIRED and RECOMMENDED to use a BCF file for performance.

--bam, -b [in.bam|in.sam|in.cram]

Sequence data in BAM/SAM/CRAM format sorted by chromosome and then position. One sample per BAM file. REQUIRED.

--ind, -i sample_name

Sample name in the VCF corresponding to the BAM file. REQUIRED.

--out, -o output

Output prefix. This will generate output.ase and output.ref_bias files. Or if you give output.gz or output.bz2 then output.ase.gz etc. REQUIRED.

--mapq, -q integer

Minimum mapping quality for a read or read pair to be considered. Set this to only include uniquely mapped reads. REQUIRED.

--fasta, -f genome_sequence.fa

Genome sequence in FASTA format. Make sure this the sequence for the correct genome build. RECOMMENDED.

--blacklist, -B poor_mappability_regions.bed

Poor mappability regions in BED format. ASE estimates will be unreliable in regions with low mappability. RECOMMENDED.

--merge-on-the-fly, -t

Merges the blacklisted regions on the fly. This can reduce memory usage if there are many overlapping or contiguous regions in the blacklist.

--gtf, -g gene_annotation.gtf

Gene annotations in GTF format. These can be obtained from <https://www.gencodegenes.org/>. RECOMMENDED.

--filtered, -l filename

File to output filtered variants. RECOMMENDED for troubleshooting especially if --suppress-warnings.

--reg, -r chr:start-end

Genomic region to be processed. E.g. chr4:12334456-16334456, or chr5

--fix-chr, -F

Attempt to match chromosome names to the BAM file by adding or removing chr to chromosome names. Does not apply to --{include,exclude}-positions options. These should be in the VCF chromosome names.

--fix-id, -R

Convert missing VCF variant IDs to chr_pos_refalt.

--auto-flip, -x

Attempt to fix reference allele mismatches. Requires a fasta file for the reference sequence by --fasta. NOT RECOMMENDED.

--print-stats, -P

Print out stats for the filtered reads for ASE sites. This will be slower and if there are sites with more reads than --max-depth, then will potentially give different results.

--suppress-warnings, -k

Suppress the warnings about individual variants.

--illumina13, -j

Base quality is in the Illumina-1.3+ encoding.

--group-by, -G integer

Group variants separated by this much into batches. This allows you not to stream the whole BAM file and may improve running time.

--max-depth, -d integer

Pileup max-depth DEFAULT=1000000. Set to more reasonable value if you experience memory or performance issues. This is set to a high value by default since with RNA-seq you can have very high coverage sites. Set to 0 for max.

--baseq, -Q integer

Minimum phred quality for a base to be considered DEFAULT=10.

--pvalue, -p float

Binomial p-value threshold for ASE output DEFAULT=1.0.

--cov, -c integer

Minimum coverage for a genotype to be considered in ASE analysis DEFAULT=16.

--cov-bias, -C integer

Minimum coverage for a genotype to be considered in reference allele mapping bias analysis DEFAULT=10.

--sites, -s integer

Minimum number of sites to calculate a reference allele mapping bias from for a specific REF/ALT pair DEFAULT=200. The reference allele mapping bias for pairs with less than this many sites will be calculated from all sites.

--imp-qual-id, -I string

The VCF INFO field ID of the imputation score in the VCF DEFAULT=INFO.

--geno-prob-id, -L string

The VCF FORMAT field ID of the genotype posterior probabilities for RR/RA/AA in the VCF DEFAULT=GP.

--imp-qual, -W float

Minimum imputation score for a variant to be considered DEFAULT=0.0.

--geno-prob, -V float

Minimum posterior probability for a genotype to be considered DEFAULT=0.0.

--subsample, -S float

Randomly subsample sites that have greater coverage than this percentile of all the sites in reference allele mapping bias calculations DEFAULT=0.75. Set to 1 to turn off which is NOT RECOMMENDED. Set to 0 to subsample all sites to --cov-bias.

--both-alleles-seen, -a

Require both alleles to be observed in RNA-seq reads for a site for ASE calculations.

--keep-bans-for-bias, -A

DON’T require both alleles to be observed in RNAseq reads for a site for reference allele mapping bias calculations. NOT RECOMMENDED.

--keep-discordant-for-bias, -E

If given, sites with more discordant alleles than REF or ALT alleles will be included in the reference allele mapping bias bias calculations. NOT RECOMMENDED.

--filter-indel-reads, -D

Remove reads that contain indels.

--keep-failed-qc, -e

Keep fastq reads that fail sequencing QC (as indicated by the sequencer).

--keep-orphan-reads, -O

Keep paired end reads where one of mates is unmapped.

--check-proper-pairing, -y

If provided only properly paired reads according to the aligner will be considered.

--ignore-orientation, -X

If NOT provided only mate pairs where both mates are on the same chromosome and where the first mate is on the +ve strand and the second is on the -ve strand will be considered. NOT RECOMMENDED.

--filter-duplicates, -u

Remove reads designated as duplicate by the aligner. NOT RECOMMENDED.

--filter-supp, -m

Remove supplementary (non-linear) alignments.

--legacy-options, -J

Replicate legacy options used. NOT RECOMMENDED.

OUTPUT FILES

.ase

This file is the main ASE results file with the following columns. Columns after the 23rd column are only printed if --print-stats is provided.

.ref_bias

Details the reference allele mapping bias results, and has the following columns:

--filtered filename

This file lists the variants that were omitted from the analysis and why. The first column is the coded omission reason, followed by the variant ID. The codes and their meaning are listed in the following section.

OUTPUT FILE CODES

.ase file codes for the CONCERN column

You probably want to exclude SNPs with RM, NRA, MDTA, or MDTR concerns from your analyses as these likely have wrong genotypes. SNPs with other concern may be OK

--filtered file codes

EXAMPLES

	o		ASE analysis of a sample mapped with STAR:
			QTLtools ase --vcf multi_sample.bcf --bam sample1.bam --ind sample1 --mapq 255 --out sample1 --filtered sample1.filtered.gz --gtf gencode.v19.annotation.gtf.gz --blacklist poor_mappability_regions.bed --fasta hg19.fa --fix-chr --fix-id

SEE ALSO

QTLtools(1)

QTLtools website: <https://qtltools.github.io/qtltools>

BUGS

Please submit bugs to <https://github.com/qtltools/qtltools>

CITATION

Delaneau, O., Ongen, H., Brown, A. et al. A complete tool set for molecular QTL discovery and analysis. Nat Commun 8, 15452 (2017). <https://doi.org/10.1038/ncomms15452>

AUTHORS

Halit Ongen (halitongen@gmail.com), Olivier Delaneau (olivier.delaneau@gmail.com)