2018-01-20

RNA seqのクオリティチェックツール QoRTs

RNA-Seqは特定のバイアス、アーティファクトを受けやすく、堅牢で包括的なクオリティチェックが重要になる。とくにサンプル調製、ライブラリー作成、またはシークエンシングのエラーは、予期せぬアーティファクト、バイアスを引き起こす。適切に処理できるように、そのような問題を検出することが重要になるが、品質を自動的にテストする包括的な方法が存在しない。

QoRTsは幅広いクオリティマトリクスを生成し、インフォマティシャンが適切にクオリティチェックを行えるようサポートする。

公式サイト

QoRTs: Quality of RNA-Seq Toolset

Example Walkthrough

QoRTs/example-walkthrough.pdf at master · hartleys/QoRTs · GitHub

インストール

Githubのリリースからコンパイル済みのjarファイルをダウンロードできる。マニュアルもある。

https://github.com/hartleys/QoRTs/releases

java -jar QoRTs.jar QC -man #マニュアル

#描画するためのRのパッケージも入れておく。Rに入って
install.packages("http://hartleys.github.io/QoRTs/QoRTs_LATEST.tar.gz", repos=NULL, type="source");

r$ java -jar QoRTs.jar QC --man

Starting QoRTs v1.3.0 (Compiled Fri Oct 20 11:56:37 EDT 2017)

Starting time: (Mon Jan 22 17:49:51 JST 2018)

NAME

Version: 1.3.0 (Updated Fri Oct 20 11:56:37 EDT 2017)

USAGE

java [Java Options] -jar QoRTs.jar QC [options] infile

gtffile.gtf qcDataDir

DESCRIPTION:

This utility runs a large battery of QC / data processing tools

on a single given sam or bam file. This is the primary function

of the QoRTs utility. All analyses are run via a single pass

through the sam/bam file.

REQUIRED ARGUMENTS:

infile

The input .bam or .sam file of aligned sequencing reads. Or

'-' to read from stdin.

(String)

gtffile.gtf

The gtf annotation file. This tool was designed to use the

standard gtf annotations provided by Ensembl, but other

annotations can be used as well.

If the filename ends with ".gz" or ".zip", the file will be

parsed using the appropriate decompression method.

(String)

qcDataDir

The output directory.

(String)

OPTIONS:

--singleEnded

Flag to indicate that reads are single end.

(flag)

--stranded

Flag to indicate that data is stranded.

(flag)

--stranded_fr_secondstrand

Flag to indicate that reads are from a fr_secondstrand type

of stranded library (equivalent to the "stranded = yes"

option in HTSeq or the "fr_secondStrand" library-type option

in TopHat/CuffLinks). If your data is stranded, you must

know the library type in order to analyze it properly. This

utility uses the same definitions as cufflinks to define

strandedness type. By default, the fr_firststrand library

type is assumed for all stranded data (equivalent to the

"stranded = reverse" option in HTSeq).

(flag)

--maxReadLength len

Sets the maximum read length. For unclipped datasets this

option is not necessary since the read length can be

determined from the data. By default, QoRTs will attempt to

determine the max read length by examining the first 1000

reads. If your data is hard-clipped prior to alignment, then

it is strongly recommended that this option be included, or

else an error may occur. Note that hard-clipping data prior

to alignment is generally not recommended, because this

makes it difficult (or impossible) to determine the

sequencer read-cycle of each nucleotide base. This may

obfuscate cycle-specific artifacts, trends, or errors, the

detection of which is one of the primary purposes of QoRTs!

In addition, hard clipping (whether before or after

alignment) removes quality score data, and thus quality

score metrics may be misleadingly optimistic. A MUCH

preferable method of removing undesired sequence is to

replace such sequence with N's, which preserves the quality

score and the sequencer cycle information while still

removing undesired sequence.

(Int)

--minMAPQ num

Filter out reads with less than the given MAPQ. Most RNA-Seq

aligners use the MAPQ field to differentiate uniquely-mapped

and multi-mapped reads. However, different aligners use a

different MAPQ conventions. By default, all reads with a

MAPQ of less than 255 will be excluded, as this is the MAPQ

associated with uniquely-aligned reads generated by the

RNA-STAR aligner. For use with TopHat2 you should set this

to 50. The MAPQ behavior for GSNAP is not well documented,

but it appears that a filtering threshold of 30 should be

adequate. Set this to 0 to turn off mapq filtering.

(Int)

--generatePlots

Generate all single-replicate QC plots. Equivalent to the

combination of: --generateMultiPlot --generateSeparatePlots

and --generatePdfReport. This option will cause QoRTs to

make an Rscript system call, loading the R package QoRTs.

(Note: this requires that R be installed and in the PATH,

and that QoRTs be installed on that R installation)

(flag)

--testRun

Flag to indicate that only the first 100k reads should be

read in. Used for testing.

(flag)

--keepMultiMapped

Flag to indicate that the tool should NOT filter out

multi-mapped reads. Note that even with this flag raised

this utility will still only use the 'primary' alignment

location for each read. By default any reads that are marked

as multi-mapped will be ignored entirely. Most aligners use

the MAPQ value to mark multi-mapped reads. Any read with

MAPQ < 255 is assumed to be non-uniquely mapped (this is the

standard used by RNA-STAR and TopHat/TopHat2). This option

is equivalent to "--minMAPQ 0".

(flag)

--noGzipOutput

Flag to indicate that output files should NOT be compressed

into the gzip format. By default almost all output files are

compressed to save space.

(flag)

--readGroup readGroupName

If this option is set, all analyses will be restricted to

ONLY reads that are tagged with the given readGroupName

(using an RG tag). This can be used if multiple read-groups

have already been combined into a single bam file, but you

want to summarize each read-group separately.

(String)

--dropChrom dropChromosomes

A comma-delimited list of chromosomes to ignore and exclude

from all analyses. Important: no whitespace!

(CommaDelimitedListOfStrings)

--skipFunctions func1,func2,...

A list of functions to skip (comma-delimited, no

whitespace). See the sub-functions list, below. The

default-on functions are: NVC, GCDistribution, GeneCalcs,

readLengthDistro, QualityScoreDistribution,

writeJunctionSeqCounts, writeKnownSplices,

writeNovelSplices, writeClippedNVC, CigarOpDistribution,

overlapMatch, cigarLocusCounts, InsertSize, chromCounts,

writeSpliceExon, writeGenewiseGeneBody, JunctionCalcs,

writeGeneCounts, writeBiotypeCounts, writeDESeq,

writeDEXSeq, writeGeneBody, StrandCheck

(CommaDelimitedListOfStrings)

--addFunctions func1,func2,...

A list of functions to add (comma-delimited, no whitespace).

This can be used to add functions that are off by default.

Followed by a comma delimited list, with no internal

whitespace. See the sub-functions list, below. The

default-off functions are: mismatchEngine,

annotatedSpliceExonCounts, calcOnTarget, FPKM, cigarMatch,

testDataDump, writeGeneBodyIv, fastqUtils, referenceMatch,

writeDocs, makeJunctionBed, makeWiggles,

makeAllBrowserTracks, calcDetailedGeneCounts

(CommaDelimitedListOfStrings)

--runFunctions func1,func2,...

The complete list of functions to run (comma-delimited, no

whitespace). Setting this option runs ONLY for the functions

explicitly requested here (along with any functions upon

which the assigned functions are dependent). See the

sub-functions list, below. Allowed options are: NVC,

mismatchEngine, annotatedSpliceExonCounts, GCDistribution,

calcOnTarget, GeneCalcs, FPKM, readLengthDistro, cigarMatch,

QualityScoreDistribution, testDataDump,

writeJunctionSeqCounts, writeKnownSplices,

writeNovelSplices, writeClippedNVC, CigarOpDistribution,

overlapMatch, cigarLocusCounts, InsertSize, chromCounts,

writeGeneBodyIv, fastqUtils, writeSpliceExon,

referenceMatch, writeGenewiseGeneBody, JunctionCalcs,

writeGeneCounts, writeDocs, makeJunctionBed,

writeBiotypeCounts, writeDESeq, writeDEXSeq, makeWiggles,

writeGeneBody, StrandCheck, makeAllBrowserTracks,

calcDetailedGeneCounts

(CommaDelimitedListOfStrings)

--seqReadCt val

(Optional) The total number of reads (or read-pairs, for

paired-end data) generated by the sequencer for this sample,

prior to alignment. This will be passed on into the

QC.summary.txt file and used to calculate mapping rate.

(Int)

--rawfastq myfastq.1.fq.gz,myfastq.2.fq.gz

(Optional) The raw fastq, prior to alignment. In normal

operation, this is used ONLY to calculate the number of

pre-alignment reads (or read-pairs) simply by counting the

number of lines and dividing by 4. Alternatively, the number

of pre-alignment read-pairs can be included explicitly via

the --seqReadCt option, or added in the plotting /

cross-comparison step by including the input.read.pair.count

column in the replicate decoder.In general, the --seqReadCt

option is recommended when available.

Certain optional QC functions are also available that

utilize the raw Fastq file in other ways. If the filename

ends with ".gz" or ".zip", the file will be parsed using the

appropriate decompression method.

(CommaDelimitedListOfStrings)

--chromSizes chrom.sizes.txt

A chrom.sizes file. The first (tab-delimited) column must

contain all chromosomes found in the dataset. The second

column must contain chromosome sizes (in base-pairs). If a

standard genome is being used, it is strongly recommended

that this be generated by the UCSC utility

'fetchChromSizes'.

This file is ONLY needed to produce wiggle files. If this is

provided, then by default QoRTs will produce 100-bp-window

wiggle files (and junction '.bed' files) for the supplied

data.In order to produce wiggle files, this parameter is

REQUIRED.

(String)

--title myTitle

The title of the replicate. Used for the track name in the

track definition line of any browser tracks ('.wig' or

'.bed' files) generated by this utility. Also may be used in

the figure text, if figures are being generated.Note that no

browser tracks will be created by default, unless the

'--chromSizes' option is set. Bed files can also be

generated using the option '--addFunction makeJunctionBed'

(String)

--flatgff flattenedGffFile.gff.gz

A "flattened" gff file that matches the standard gtf file.

Optional. The "flattened" gff file assigns unique

identifiers for all exons, splice junctions, and

aggregate-genes. This is used for the junction counts and

exon counts (for DEXSeq). The flattened gtf file can be

generated using the "makeFlatGff" command. Flattened GFF

files containing novel splice junctions can be generated

using the "mergeNovelSplices" function. Note that (for most

purposes) the command should be run with the same

strandedness code as found in the dataset. Running a

flattened gff that was generated using a different

strandedness mode may be useful for certain purposes, but is

generally not supported and is for advanced users only.See

the documentation for makeFlatGff for more information.

If the filename ends with ".gz" or ".zip", the file will be

parsed using the appropriate decompression method.

(String)

--generateMultiPlot

Generate a multi-frame figure, containing a visual summary

of all QC stats. (Note: this requires that R be installed

and in the PATH, and that QoRTs be installed on that R

installation)

(flag)

--generateSeparatePlots

Generate seperate plots for each QC stat, rather than only

one big multiplot. (Note: this requires that R be installed

and in the PATH, and that QoRTs be installed on that R

installation)

(flag)

--generatePdfReport

Generate a pdf report. (Note: this requires that R be

installed and in the PATH, and that QoRTs be installed on

that R installation)

(flag)

--adjustPhredScore val

QoRTs expects input files to conform to the SAM format

specification, which requires all Phred scores to be in

Phred+33 encoding. However some older tools produce SAM

files with nonstandard encodings. To read such data, you can

set this parameter to subtract from the apparent (phred+33)

phred score. Thus, to read Phred+64 data (produced by

Illumina v1.3-1.7), set this parameter to 31. Note: QoRTs

does not support negative Phred scores. NOTE: THIS OPTION IS

EXPERIMENTAL!

(Int)

--maxPhredScore val

According to the standard FASTQ and SAM format

specification, Phred quality scores are supposed to range

from 0 to 41. However, certain sequencing machines such as

the HiSeq4000 supposedly produce occasional quality scores

as high as 45. If your dataset contains quality scores in

excess of 41, then you must use this option to set the

maximum legal quality score. Otherwise, QoRTs will throw an

error.

(Int)

--summaryFileSuffix .summary.txt

The suffix of the 'summary' file. This is useful to set if

you want to run multiple QC runs in parallel to reduce

runtime, without overwriting one another's summary files.In

particular, the NVC metrics often take a long time to run,

so splitting those off using the --runFunctions parameter

might speed things up considerably. Note that 'QC' will be

appended in the actual filename. THIS OPTION IS BETA!

(String)

--extractReadsByMetric metric=value

THIS OPTIONAL PARAMETER IS STILL UNDER BETA TESTING. This

parameter allows the user to extract anomalous reads that

showed up in previous QoRTs runs. Currently reads can be

extracted based on the following metrics: StrandTestStatus,

InsertSize and GCcount.

(String)

--keepOnlyOnTarget

Experimental flag. Ignores reads that DO NOT fall within the

target region (specified by the required bedfile using the

--targetRegionBed parameter).

(flag)

--dropOnTarget

Experimental flag. Ignores reads that DO fall within the

target region (specified by the required bedfile using the

--targetRegionBed parameter).

(flag)

--randomSubsample 1.00

If this option is set, QoRTs will ignore a random fraction

of the input read pairs. This can drastically reduce

runtime, though it may reduce the accuracy of the output QC

metrics.

(Double)

--restrictToGeneList geneList.txt

If this option is set, almost all analyses will be

restricted to reads that are found on genes named in the

supplied gene list file. The file should contain a gene ID

on each line and nothing else. The only functions that will

be run on the full set of all reads will be the functions

that calculate the gene mapping itself. NOTE: if you want to

include ambiguous reads, include a line with the text:

'_ambiguous'. If you want to include reads that do not map

to any known feature, include a line with the text:

'_no_feature'. WARNING: this is not intended for default

use. It is intended to be used when re-running QoRTs, with

the intention of examining artifacts that can be caused in

various plots by a small number of genes with extremely high

coverage. For example, GC content plots sometimes contain

visible spikes caused by small mitochondrial genes with

extremely high expression.ADDITIONAL WARNING: This feature

is in BETA, and is not yet fully tested.

(String)

--dropGeneList geneList.txt

If this option is set, almost all analyses will be

restricted to reads that are NOT found on genes named in the

supplied gene list file. The file should contain a gene ID

on each line and nothing else. The only functions that will

be run on the full set of all reads will be the functions

that calculate the gene mapping itself. NOTE: if you want to

EXCLUDE ambiguous reads, include a line with the text:

'_ambiguous'. If you want to EXCLUDE reads that do not map

to any known feature, include a line with the text:

'_no_feature'. WARNING: this is not intended for default

use. It is intended to be used when re-running QoRTs, with

the intention of examining artifacts that can be caused by

certain individual 'problem genes'. For example, GC content

plots sometimes contain visible spikes caused by small

transcripts / RNA's with extremely high expression

levels.ADDITIONAL WARNING: This feature is in BETA, and is

not yet fully tested.

(String)

--DNA

BETA: This flag makes various changes to allow QoRTs to run

on whole-exome or whole-genome DNA-Seq data.

(flag)

--RNA

Indicates that the data is RNA-Seq (this is the default:

flag does nothing).

(flag)

--genomeFA chr.fa.gz[,chr2.fa,...]

Reference genome sequence. This can either be a single FASTA

file with all the chromosomes included, or a comma-delimited

list of fasta files with 1 chromosome each. Note: IF

multiple fasta files are specificed, each must contain ONLY

ONE chromosome. If a single multi-chromosome fasta file is

specificed, performance will be improved if the chromosomes

are in the same order as they are found in the BAM file,

however, this is not required. The genomic sequence is used

by certain experimental sub-utilities (currently only the

referenceMatch utility). Comma delimited, no spaces. Fasta

files can be in plaintext, gzipped or zipped.

(CommaDelimitedListOfStrings)

--genomeBufferSize val

The size of the genome fasta buffer. Tuning this parameter

may improve performance.

(Int)

--outfilePrefix sampID

Prefix to be prepended to all output files. If this is set,

all output files will use the format:

"outfiledir/<prefix>QC.qcfilename.txt.gz"

(String)

--nameSorted

DEPRECATED: Relevant for paired-end reads only.

This flag is used to run QoRTs in "name-sorted" mode. This

flag is optional, as under the default mode QoRTs will

accept BAM files sorted by either name OR position. However,

The only actual requirement in this mode is that read pairs

be adjacent.

Errors may occur if the SAM flags are inconsistent: for

example, if orphaned reads appear with the "mate mapped" SAM

flag set.

(flag)

--coordSorted

DEPRECATED: this mode is now subsumed by the default mode

and as such this parameter is now nonfunctional.

Note that, in the default mode, for paired-end data QoRTs

will accept EITHER coordinate-sorted OR name-sorted bam

files. In "--nameSorted" mode, QoRTs ONLY accepts

name-sorted bam files.

If a large fraction of the read-pairs are mapped to

extremely distant loci (or to different chromosomes), then

memory issues may arise. However, this should not be a

problem with most datasets. Technically by default QoRTs can

run on arbitrarily-ordered bam files, but this is STRONGLY

not recommended, as memory usage will by greatly increased.

(flag)

--fileContainsNoMultiMappedReads

DEPRECATED. Flag to indicate that the input sam/bam file

contains only primary alignments (ie, no multi-mapped

reads). This flag is ALWAYS OPTIONAL, but when applicable

this utility will run (slightly) faster when using this

argument. (DEPRECIATED! The performance improvement was

marginal)

(flag)

--parallelFileRead

DEPRECATED: DO NOT USE. Flag to indicate that bam file

reading should be run in paralell for increased speed. Note

that in this mode you CANNOT read from stdin. Also note that

for this to do anything useful, the numThreads option must

be set to some number greater than 1. Also note that

additional threads above 9 will have no appreciable affect

on speed.

(flag)

--numThreads num

DEPRECIATED, nonfunctional.

(Int)

--checkForAlignmentBlocks

Certain aligners will mark reads 'aligned' even though they

have no aligned bases. This option will automatically check

for some reads and ignore them, rather than throwing an

error.

(flag)

--targetRegionBed targetRegion.bed

For whole exome sequencing, this specifies the exome target

regions.

(String)

--stopAfterNReads n

Stop after reading in n reads or read-pairs.

(Int)

--randomSeed n

Use specified random seed.

(Long)

--parseIlluminaStyleReadIDs

Specifies that the read-names are in the illumina style.

CURRENTLY NONFUNCTIONAL!

(flag)

--verbose

Flag to indicate that debugging information and extra

progress information should be sent to stderr.

(flag)

--quiet

Flag to indicate that only errors and warnings should be

sent to stderr.

(flag)

DEFAULT SUB-FUNCTIONS

NVC

Nucleotide-vs-Cycle counts.

GCDistribution

Calculate GC content distribution.

GeneCalcs

Find gene assignment and gene body calculations.

readLengthDistro

Tabulates the distribution of read lengths.

QualityScoreDistribution

Calculate quality scores by cycle.

writeJunctionSeqCounts

Write counts file designed for use with JunctionSeq

(contains known splice junctions, gene counts, and exon

counts). [Depends: writeSpliceExon]

writeKnownSplices

Write known splice junction counts. [Depends: JunctionCalcs]

writeNovelSplices

Write novel splice junction counts. [Depends: JunctionCalcs]

writeClippedNVC

Write NVC file containing clipped sequences. [Depends: NVC]

CigarOpDistribution

Cigar operation rates by cycle and cigar operation length

rates (deletions, insertions, splicing, clipping, etc).

overlapMatch

BETA: This function calculates the matching of overlapping

sections of paired reads. [Depends: mismatchEngine]

cigarLocusCounts

BETA: This function is still undergoing basic testing. It is

not intended for production use at this time.

InsertSize

Insert size distribution (paired-end data only).

chromCounts

Calculate chromosome counts

writeSpliceExon

Synonym for function "writeJunctionSeqCounts" (for

backwards-compatibility) [Depends: JunctionCalcs]

writeGenewiseGeneBody

Write file containing gene-body distributions for each

(non-overlapping) gene. [Depends: writeGeneBody]

JunctionCalcs

Find splice junctions (both novel and annotated).

writeGeneCounts

Write extended gene-level read/read-pair counts file

(includes counts for CDS/UTR, ambiguous regions, etc).

[Depends: GeneCalcs]

writeBiotypeCounts

Write a table listing read counts for each gene BioType

(uses the optional "gene_biotype" GTF attribute). [Depends:

GeneCalcs]

writeDESeq

Write gene-level read/read-pair counts file, suitable for

use with DESeq, EdgeR, etc. [Depends: GeneCalcs]

writeDEXSeq

Write exon-level read/read-pair counts file, designed for

use with DEXSeq. [Depends: JunctionCalcs]

writeGeneBody

Write gene-body distribution file. [Depends: GeneCalcs]

StrandCheck

Check the strandedness of the data. Note that if the

stranded option is set incorrectly, this tool will

automatically print a warning to that effect.

NON-DEFAULT SUB-FUNCTIONS

mismatchEngine

Internal module that runs overlap/reference mismatch

calculations. Automatically included on any runs that

include these functions.

annotatedSpliceExonCounts

Write counts for exons, known-splice-junctions, and genes,

with annotation columns indicating chromosome, etc (default:

OFF) [Depends: JunctionCalcs]

calcOnTarget

BETA: requires --targetRegionBed parameter. This function

calculates the rates at which reads intersect with the

On-Target area. Intended for whole exome sequencing data.

Make sure to use the --targetRegionBed parameter or else

this function will deactivate! (Default: ON iff

targetRegionBed param is found)

FPKM

Write FPKM values. Note: FPKMs are generally NOT the

recommended normalization method. We recommend using a more

advanced normalization as provided by DESeq, edgeR,

CuffLinks, or similar (default: OFF)

cigarMatch

Work-In-Progress: this function is a placeholder for future

functionality, and is not intended for use at this time.

(default: OFF)

testDataDump

EXPERIMENTAL: This function dumps a bunch of information for

internal testing purposes. NOT FOR GENERAL USE! (default:

OFF)

writeGeneBodyIv

Writes an optional additional file detailing the intervals

used in the gene-body coverage calculations

("QC.geneBodyCoverage.DEBUG.intervals.txt.gz") (default:

OFF) [Depends: writeGeneBody]

fastqUtils

BETA: requires --rawfastq parameter. Adds additional tests

that use the supplied raw fastq file. Requires that one (or

two) fastq files be supplied. (Default: ON iff rawfastq

param is found)

referenceMatch

BETA: requires --genomeFA parameter. This function

calculates the matching against the reference. Requires the

specification of genome fasta file(s). REQUIRES

COORDINATE-SORTED BAM FILES! REQUIRES THAT FA AND BAM HAVE

THE SAME CHROMOSOME ORDERING! (Default: ON iff genomeFA

param is found) [Depends: mismatchEngine]

writeDocs

Writes a QC.documentation.txt file that documents all output

files.

makeJunctionBed

Write splice-junction count "bed" files. (default: OFF)

makeWiggles

Write "wiggle" coverage files with 100-bp window size. Note:

this REQUIRES that the --chromSizes parameter be included!

(default: OFF)

makeAllBrowserTracks

Write both the "wiggle" and the splice-junction bed files

(default: OFF) [Depends: makeJunctionBed, makeWiggles]

calcDetailedGeneCounts

Calculate more detailed read counts for each gene, counting

the number of reads that cover introns, cross-strand, etc

(default: OFF)

AUTHORS:

Stephen W. Hartley, Ph.D. stephen.hartley (at nih dot gov)

LEGAL:

This software is "United States Government Work" under the terms

of the United States Copyright Act. It was written as part of

the authors' official duties for the United States Government

and thus cannot be copyrighted. This software is freely

available to the public for use without a copyright notice.

Restrictions cannot be placed on its present or future use.

Although all reasonable efforts have been taken to ensure the

accuracy and reliability of the software and data, the National

Human Genome Research Institute (NHGRI) and the U.S. Government

does not and cannot warrant the performance or results that may

be obtained by using this software or data. NHGRI and the U.S.

Government disclaims all warranties as to performance,

merchantability or fitness for any particular purpose.

In any work or product derived from this material, proper

attribution of the authors as the source of the software or data

should be made, using "NHGRI Genome Technology Branch" as the

citation.

NOTE: This package includes (internally) the sam-1.113.jar

library from picard tools. That package uses the MIT license,

which can be accessed using the command:

java -jar thisjarfile.jar help samjdkinfo

Done. (Mon Jan 22 17:49:51 JST 2018)

例えばbin/に入れて省略名"QoRTs"でランできるようにしておく。

mv QoRTs.jar /usr/local/bin/
echo alias QoRTs=\'java -jar /usr/local/bin/QoRTs.jar\' >> ~/.bash_profile && source ~/.bash_profile

ラン

アライメント結果のbamとgtfを指定してランする。結果は指定したディレクトリに出力される。

QoRTs --generatePdfReport input.bam input.gtf qcDataDir

--singleEnded　Flag to indicate that reads are single end. (flag)
--stranded　Flag to indicate that data is stranded. (flag)
--stranded_fr_secondstrand　Flag to indicate that reads are from a fr_secondstrand type of stranded library (equivalent to the "stranded = yes" option in HTSeq or the "fr_secondStrand" library-type option in TopHat/CuffLinks). If your data is stranded, you must know the library type in order to analyze it properly. This utility uses the same definitions as cufflinks to define strandedness type. By default, the fr_firststrand library type is assumed for all stranded data (equivalent to the "stranded = reverse" option in HTSeq). (flag)
--generateMultiPlot　Generate a multi-frame figure, containing a visual summary of all QC stats. (Note: this requires that R be installed and in the PATH, and that QoRTs be installed on that R installation) (flag)
--generatePdfReport　Generate a pdf report. (Note: this requires that R be installed and in the PATH, and that QoRTs be installed on that R installation)

シングルエンドのbamなら"--singleEnded"のフラグをつけてランする。結果は指定したディレクトリqcDataDir/に出力される。

--generatePdfReportをつけると、次のようなPDFレポートが出力される。

f:id:kazumaxneo:20180122180004j:plain