FastQCの高速な代替 Falco - macでインフォマティクス

　品質管理はシーケンスデータ解析において不可欠な最初のステップであり、品質管理のためのソフトウェアツールはほとんどのシーケンスセンターで標準的なパイプラインに深く浸透している。関連する計算は簡単だが、多くの環境では品質管理に必要な総計算量はかなりのものであり、最適化が必要である。Falcoは、FastQCのエミュレーションであり、同等の結果を生成しながら平均3倍速く動作する。FastQCと比較して、Falcoは実行に必要なメモリも少なく、HTMLレポートのより柔軟な視覚化を提供する。

インストール

ubuntu20とmacでテストした。

依存

zlib is required to read gzip compressed FASTQ files
htslib is required to process bam files

Github

https://github.com/smithlabcode/falco

#conda
mamba install -c bioconda falco -y

#source
git clone https://github.com/smithlabcode/falco.git
cd falco/
./autogen.sh
./configure CXXFLAGS="-O3 -Wall" --enable-hts
make HAVE_HTSLIB=1 all
make HAVE_HTSLIB=1 install

> falco -h

Usage: falco [OPTIONS] <seqfile1> <seqfile2> ...

Options:

-h, --help Print this help file and exit

-v, --version Print the version of the program and exit

-o, --outdir Create all output files in the specified

output directory. FALCO-SPECIFIC: If the

directory does not exists, the program will

create it. If this option is not set then

the output file for each sequence file is

created in the same directory as the

sequence file which was processed.

--casava [IGNORED BY FALCO] Files come from raw

casava output. Files in the same sample

group (differing only by the group number)

will be analysed as a set rather than

individually. Sequences with the filter flag

set in the header will be excluded from the

analysis. Files must have the same names

given to them by casava (including being

gzipped and ending with .gz) otherwise they

won't be grouped together correctly.

--nano [IGNORED BY FALCO] Files come from nanopore

sequences and are in fast5 format. In this

mode you can pass in directories to process

and the program will take in all fast5 files

within those directories and produce a

single output file from the sequences found

in all files.

--nofilter [IGNORED BY FALCO] If running with --casava

then don't remove read flagged by casava as

poor quality when performing the QC

analysis.

--extract [ALWAYS ON IN FALCO] If set then the zipped

output file will be uncompressed in the same

directory after it has been created. By

default this option will be set if fastqc is

run in non-interactive mode.

-j, --java [IGNORED BY FALCO] Provides the full path to

the java binary you want to use to launch

fastqc. If not supplied then java is assumed

to be in your path.

--noextract [IGNORED BY FALCO] Do not uncompress the

output file after creating it. You should

set this option if you do not wish to

uncompress the output when running in

non-interactive mode.

--nogroup Disable grouping of bases for reads >50bp.

All reports will show data for every base in

the read. WARNING: When using this option,

your plots may end up a ridiculous size. You

have been warned!

--min_length [NOT YET IMPLEMENTED IN FALCO] Sets an

artificial lower limit on the length of the

sequence to be shown in the report. As long

as you set this to a value greater or equal

to your longest read length then this will

be the sequence length used to create your

read groups. This can be useful for making

directly comaparable statistics from

datasets with somewhat variable read

lengths.

-f, --format Bypasses the normal sequence file format

detection and forces the program to use the

specified format. Validformats are bam, sam,

bam_mapped, sam_mapped, fastq, fq, fastq.gz

or fq.gz.

-t, --threads [NOT YET IMPLEMENTED IN FALCO] Specifies the

number of files which can be processed

simultaneously. Each thread will be

allocated 250MB of memory so you shouldn't

run more threads than your available memory

will cope with, and not more than 6 threads

on a 32 bit machine [1]

-c, --contaminants Specifies a non-default file which contains

the list of contaminants to screen

overrepresented sequences against. The file

must contain sets of named contaminants in

the form name[tab]sequence. Lines prefixed

with a hash will be ignored. Default:

/home/kazu/Documents/falco/Configuration/contaminant_list.txt

-a, --adapters Specifies a non-default file which contains

the list of adapter sequences which will be

explicity searched against the library. The

file must contain sets of named adapters in

the form name[tab]sequence. Lines prefixed

with a hash will be ignored. Default:

/home/kazu/Documents/falco/Configuration/adapter_list.txt

-l, --limits Specifies a non-default file which contains

a set of criteria which will be used to

determine the warn/error limits for the

various modules. This file can also be used

to selectively remove some modules from the

output all together. The format needs to

mirror the default limits.txt file found in

the Configuration folder. Default:

/home/kazu/Documents/falco/Configuration/limits.txt

-k, --kmers [IGNORED BY FALCO AND ALWAYS SET TO 7]

Specifies the length of Kmer to look for in

the Kmer content module. Specified Kmer

length must be between 2 and 10. Default

length is 7 if not specified.

-q, --quiet Supress all progress messages on stdout and

only report errors.

-d, --dir [IGNORED: FALCO DOES NOT CREATE TMP FILES]

Selects a directory to be used for temporary

files written when generating report images.

Defaults to system temp directory if not

specified.

-s, -subsample [Falco only] makes falco faster (but

possibly less accurate) by only processing

reads that are multiple of this value (using

0-based indexing to number reads). [1]

-b, -bisulfite [Falco only] reads are whole genome

bisulfite sequencing, and more Ts and fewer

Cs are therefore expected and will be

accounted for in base content.

-r, -reverse-complement [Falco only] The input is a

reverse-complement. All modules will be

tested by swapping A/T and C/G

-skip-data [Falco only] Do not create FastQC data text

file.

-skip-report [Falco only] Do not create FastQC report

HTML file.

-skip-summary [Falco only] Do not create FastQC summary

file

-D, -data-filename [Falco only] Specify filename for FastQC

data output (TXT). If not specified, it will

be called fastq_data.txt in either the input

file's directory or the one specified in the

--output flag. Only available when running

falco with a single input.

-R, -report-filename [Falco only] Specify filename for FastQC

report output (HTML). If not specified, it

will be called fastq_report.html in either

the input file's directory or the one

specified in the --output flag. Only

available when running falco with a single

input.

-S, -summary-filename [Falco only] Specify filename for the short

summary output (TXT). If not specified, it

will be called fastq_report.html in either

the input file's directory or the one

specified in the --output flag. Only

available when running falco with a single

input.

-K, -add-call [Falco only] add the command call call to

FastQC data output and FastQC report HTML

(this may break the parse of fastqc_data.txt

in programs that are very strict about the

FastQC output format).

Help options:

-?, -help print this help message

-about print about message

実行方法

fastqファイルを指定する（fastq, fq, fastq.gz or fq.gz）。

#１つのfastq
falco input.fq.gz

#複数fastq、#出力指定、4スレッド
falco -t 4 -o outdir sample*.fq.gz

-o Create all output files in the specified output directory. FALCO-SPECIFIC: If the directory does not exists, the program will create it. If this option is not set then the output file for each sequence file is created in the same directory as the sequence file which was processed.
-t Specifies the number of files which can be processed simultaneously. Each thread will be allocated 250MB of memory so you shouldn't run more threads than your available memory will cope with, and not more than 6 threads on a 32 bit machine [1]
-c Specifies a non-default file which contains the list of contaminants to screen overrepresented sequences against. The file must contain sets of named contaminants in the form name[tab]sequence. Lines prefixed with a hash will be ignored. Default: Configuration/contaminant_list.txt
-a Specifies a non-default file which contains the list of adapter sequences which will be explicity searched against the library. The file must contain sets of named adapters in the form name[tab]sequence. Lines prefixed with a hash will be ignored. Default: Configuration/adapter_list.txt

sam、bam (binary sam)のリードの分析にも対応している（htslibが必要）。

falco -t 4 -o outdir input.bam

-f Bypasses the normal sequence file format detection and forces the program to use the specified format. Valid formats are bam, sam, bam_mapped, sam_mapped, fastq, fq, fastq.gz or fq.gz.

出力