Provided by: qtltools_1.3.1+dfsg-2build2_amd64 
NAME
QTLtools quan - Quantify gene and exon expression from RNA-seq
SYNOPSIS
QTLtools quan --bam [in.sam|in.bam|in.cram] --gtf gene_annotation.gtf --out-prefix output
[OPTIONS]
DESCRIPTION
This mode quantifies the expression of genes and exons in the provided --gtf file using
the RNA-seq reads in the --bam file. The method counts the number of reads overlapping
the exons in the --gtf file. Firstly all exons of a gene are converted into meta-exons
where overlapping exons are merged into a single exon encompassing all the overlapping
exons. Any overlap between the read and the exon is considered a match, that is a read is
not required to be in between start and end positions of an exon to count towards that
exon's quantification. Split reads aligning to multiple exons contribute to each exon it
overlaps with based on the fraction of the read that overlaps with a given exon. Thus
split reads contribute less than a single count to each of the overlapping exons. Reads
aligning to multiple exons (i.e. overlapping exons of multiple genes) count towards the
quantification of all the exons that it overlaps with. If the --bam file contains paired-
end reads and if there are cases where the two mate pairs overlap with each other (i.e.
have an insert size < 0), then each of these reads contribute less then a single count
towards the quantifications unless --no-merge is provided. The following diagram, with
two genes with overlapping exons and one paired-end read where both mate pairs are split
reads and overlap with each other, illustrates how the quantification works:
x x
/ \ / \
+---------+ +---------+ +---------+
| Exon1|1 | | Exon1|2 | | Exon1|3 | Gene1
+---------+ +---------+ +---------+
x
/ \
+------------+ +-------------+
| Exon2|1 | | Exon2|2 | Gene2
+------------+ +-------------+
x
/ \
+------------+ +----+ RNAseq Read Mate1
|--a-||-b-||c| |-d--||--e-|
x
/ \
+------+ +----------+ RNAseq Read Mate2
Left Mate1 = ((b * 0.5) + a) / (a + b + d)
Right Mate1 = (d * 0.5)/(a + b + d)
Left Mate2 = (b * 0.5)/(b + d + e)
Right Mate2 = ((d * 0.5) + e)/(b + d + e)
Exon1|2 = Left Mate1 + Left Mate2
Exon1|3 = Right Mate1 + Right Mate2
Exon2|1 = Left Mate1 + Left Mate2
Exon2|2 = Right Mate2 + Right Mate2
Gene1 = Exon1|2 + Exon1|3
Gene2 = Exon2|1 + Exon2|2
The quan mode in version 1.2 and above is not compatible with the quantifications
generated by the previous versions. This due to bug fixes and slight adjustments to the
way we quantify. DO NOT MIX QUANTIFICATIONS GENERATED BY EARLIER VERSIONS OF QTLTOOLS WITH
QUANTIFICATIONS FROM VERSION 1.2 AND ABOVE AS THIS WILL CREATE A BIAS IN YOUR DATASET.
OPTIONS
--gtf gene_annotation.gtf
Gene annotations in GTF format. These can be obtained from
<https://www.gencodegenes.org/>. REQUIRED.
--bam [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.
--out-prefix output
Output prefix. REQUIRED.
--sample sample_name
The sample name of the BAM file. If not provided the sample name will be taken as
the BAM file path.
--rpkm Output RPKM values.
--tpm Output TPM values.
--xxhash
Rather than using the GTF file name to generate unique hash for the options used,
use the hash of the GTF file.
--no-hash
Do not include a hash signifying the options used in the quantification in the
output file names. NOT RECOMMENDED.
--gene-types gene_type ...
Only quantify these gene types. Requires gene_type attribute in GTF. It will also
use transcript_type if present.
--filter-mapping-quality integer
Minimum mapping quality for a read or read pair to be considered. Set this to only
include uniquely mapped reads. DEFAULT=10.
--filter-mismatch integer|float
Maximum mismatches allowed in a read. If between 0 and 1 taken as the fraction of
read length. Requires NM attribute in the BAM file. DEFAULT=OFF.
--filter-mismatch-total integer|float
Maximum total mismatches allowed in paired-reads. If between 0 and 1 taken as the
fraction of combined read length. Requires NM attribute in the BAM file.
DEFAULT=OFF.
--filter-min-exon integer
Minimum length of an exon for it to be quantified. Exons smaller than this will
not be printed out in the exon quantifications, but will still count towards gene
quantifications. DEFAULT=0.
--filter-remove-duplicates
Remove duplicate sequencing reads,as indicated by the aligner, in the process. NOT
RECOMMENDED.
--filter-failed-qc
Remove fastq reads that fail sequencing QC as indicated by the sequencer.
--check-proper-pairing
If provided only properly paired reads according to the aligner that are in correct
orientation will be considered. Otherwise all pairs in correct orientation will be
considered.
--check-consistency
If provided checks the consistency of split reads with annotation, rather than pure
overlap of one of the blocks of the split read.
--no-merge
If provided overlapping mate pairs will not be merged. Default behavior is to
merge overlapping mate pairs based on the amount of overlap, such that each mate
pair counts for less than 1 read.
--legacy-options
Exactly replicate Dermitzakis lab original quantification script. DO NOT USE.
--region chr:start-end
Genomic region to be processed. E.g. chr4:12334456-16334456, or chr5.
OUTPUT FILES
Unless --no-hash is provided, all output files will include a hash value corresponding to
combination of the specific options used. This is given so that one does not merge
quantifications from samples that were quantified differently, which would create a bias
in the dataset.
.gene.count.bed .exon.count.bed .gene.rpkm.bed .exon.rpkm.bed .gene.tpm.bed .exon.tpm.bed
These are the quantification results files with the following columns:
1 chr Phenotype's chromosome
2 start Phenotype's start position (0-based)
3 end Phenotype's end position (1-based)
4 gene|exon The gene or exon ID.
5 info|geneID Information about the gene or the gene ID of the exon. The gene info
is separated by semicolons, and L=gene length, T=gene type, R=gene
positions, N=gene name
6 strand Phenotype's strand
7 sample_name The sample name of the BAM file
.stats
Details the statistics of the quantification, with the following rows:
1 filtered_secondary_alignments_(does_not_count_towards_total_reads) Number of
secondary
alignments
2 total_reads Number of reads
in the BAM file
3 filtered_unmapped Number of
unmapped reads
4 filtered_failqc Number of reads
with the failed
QC tag
5 filtered_duplicate Number of
duplicate reads
6 filtered_mapQ_less_than_X Number of reads
below the
mapping quality
threshold X
7 filtered_notpaired Number of pairs
that were not
in the correct
orientation or
were not
properly paired
8 filtered_mismatches_greater_than_X_Y Number of reads
failing the
mismatches per
read, X, and
mismatches
total filters,
Y
9 filtered_unmatched_mate_pairs Number of reads
where there was
a paired-read
with a missing
mate
10 total_good Number of reads
that passed all
filters
11 total_exonic Number of reads
that aligned to
exons and
passed all
filters
12 total_exonic_multi_counting Number of reads
that aligned to
exons when we
count reads
that align to
multiple exons
multiple times
13 total_merged_reads Number of reads
where the mate
pairs were
overlapping and
thus were
merged
14 total_exonic_multi_counting_after_merge_(used_for_rpkm) Number of reads
that aligned to
exons when we
merge
overlapping
mate pairs
15 good_over_total Number of good
reads over the
total number of
reads
16 exonic_over_total Number of
exonic reads
over the total
number of reads
17 exonic_over_good Number of
exonic reads
over the number
of good reads
EXAMPLE
o Quantifying a sample mapped with GEM, outputting TPM and RPKM values, and taking the
hash of the GTF file:
QTLtools quan --bam HG00381.chr22.bam --gtf gencode.v19.annotation.chr22.gtf.gz --out-
prefix HG00381 --sample HG00381 --rpkm --tpm --xxhash --filter-mismatch-total 8
--filter-mapping-quality 150
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)