trimgalore

trimming

adapters

Description

A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files.

Input

Name	Type & Properties	Description
--input	file required multiple	Input files. Note that paired-end files need to be supplied in a pairwise fashion, e.g. file1_1.fq file1_2.fq SRR2_1.fq.gz SRR2_2.fq.gz

Name

Type & Properties

--input

file

required

multiple

Trimming options

Name	Type & Properties	Description
--quality -q	integer	Trim low-quality ends (below the specified Phred score) from reads in addition to adapter removal. For RRBS samples, quality trimming will be performed first, and adapter trimming is carried in a second round. Other files are quality and adapter trimmed in a single pass. The algorithm is the same as the one used by BWA (Subtract INT from all qualities; compute partial sums from all indices to the end of the sequence; cut sequence at the index at which the sum is minimal).
--phred33	boolean_true	Instructs Cutadapt to use ASCII+33 quality scores as Phred scores (Sanger/Illumina 1.9+ encoding) for quality trimming.
--phred64	boolean_true	Instructs Cutadapt to use ASCII+64 quality scores as Phred scores (Illumina 1.5 encoding) for quality trimming.
--fastqc	boolean_true	Run FastQC in the default mode on the FastQ file once trimming is complete.
--fastqc_args	string	Passes extra arguments (excluding files) to FastQC. If more than one argument is to be passed to FastQC they must be in the form "arg1 arg2 ...". Passing extra arguments will automatically invoke FastQC, so --fastqc does not have to be specified separately.
--fastqc_contaminants	file	Specifies a non-default file which contains the list of contaminants for FastQC 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.
--fastqc_adapters	file	Specifies a non-default file which contains the list of adapter sequences which which FasstQC will explicity search 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.
--fastqc_limits	file	Specifies a non-default file which contains a set of criteria which FastQC will use 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.
--adapter -a	string	Adapter sequence to be trimmed. If not specified explicitly, Trim Galore will try to auto-detect whether the Illumina universal, Nextera transposase or Illumina small RNA adapter sequence was used. A single base may also be given as e.g. -a A{10}, to be expanded to -a AAAAAAAAAA. At a special request, multiple adapters can also be specified like so: -a " AGCTCCCG -a TTTCATTATAT -a TTTATTCGGATTTAT" -a2 " AGCTAGCG -a TCTCTTATAT -a TTTCGGATTTAT", or so: -a "file:../multiple_adapters.fa" -a2 "file:../different_adapters.fa" Potentially in conjucntion with the parameter "-n 3" to trim all adapters.
--adapter2 -a2	string	Optional adapter sequence to be trimmed off read 2 of paired-end files. This option requires '--paired' to be specified as well. If the libraries to be trimmed are smallRNA then a2 will be set to the Illumina small RNA 5' adapter automatically (GATCGTCGGACT). A single base may also be given as e.g. -a2 A{10}, to be expanded to -a2 AAAAAAAAAA.
--illumina	boolean_true	Adapter sequence to be trimmed is the first 13bp of the Illumina universal adapter 'AGATCGGAAGAGC' instead of the default auto-detection of adapter sequence.
--stranded_illumina	boolean_true	Adapter sequence to be trimmed is the first 13bp of the Illumina stranded mRNA or Total RNA adapter 'ACTGTCTCTTATA' instead of the default auto-detection of adapter sequence.
--nextera	boolean_true	Adapter sequence to be trimmed is the first 12bp of the Nextera adapter 'CTGTCTCTTATA' instead of the default auto-detection of adapter sequence.
--small_rna	boolean_true	Adapter sequence to be trimmed is the first 12bp of the Illumina Small RNA 3' Adapter 'TGGAATTCTCGG' instead of the default auto-detection of adapter sequence. Selecting to trim smallRNA adapters will also lower the --length value to 18bp. If the smallRNA libraries are paired-end then a automatically (GATCGTCGGACT) unless -a 2 had been defined explicitly.
--consider_already_trimmed	integer	During adapter auto-detection, the limit set by this argument allows the user to set a threshold up to which the file is considered already adapter-trimmed. If no adapter sequence exceeds this threshold, no additional adapter trimming will be performed (technically, the adapter is set to '-a X'). Quality trimming is still performed as usual.
--max_length	integer	Discard reads that are longer than the specified value after trimming. This is only advised for smallRNA sequencing to remove non-small RNA sequences.
--stringency	integer	Overlap with adapter sequence required to trim a sequence. Defaults to a very stringent setting of 1, i.e. even a single bp of overlapping sequence will be trimmed off from the 3' end of any read.
--error_rate -e	double	Maximum allowed error rate (no. of errors divided by the length of the matching region)
--gzip	boolean_true	Compress the output file with GZIP. If the input files are GZIP-compressed the output files will automatically be GZIP compressed as well. As of v0.2.8 the compression will take place on the fly.
--dont_gzip	boolean_true	Output files won't be compressed with GZIP. This option overrides --gzip.
--length	integer	Discard reads that became shorter than the specified length because of either quality or adapter trimming. A value of '0' effectively disables this behaviour. For paired-end files, both reads of a read-pair need to be longer than the specified length to be printed out to validated paired-end files. If only one read became too short there is the possibility of keeping such unpaired single-end reads using the --retain_unpaired option.
--max_n	integer	The total number of Ns a read may contain before it will be removed altogether.In a paired-end setting, either read exceeding this limit will result in the entire pair being removed from the trimmed output files. If COUNT is a number between 0 and 1, it is interpreted as a fraction of the read length.
--trim_n	boolean_true	Removes Ns from either side of the read. This option does currently not work in RRBS mode.
--no_report_file	boolean_true	If specified no report file will be generated.
--suppress_warn	boolean_true	If specified any output to STDOUT or STDERR will be suppressed.
--clip_R1	integer	Instructs TrimGalore to remove given number of bp from the 5' end of read 1 (or single-end reads). This may be useful if the qualities were very poor, or if there is some sort of unwanted bias at the 5' end.
--clip_R2	integer	Instructs TrimGalore to remove given number bp from the 5' end of read 2 (paired-end reads only). This may be useful if the qualities were very poor, or if there is some sort of unwanted bias at the 5' end. For paired-end BS-Seq, it is recommended to remove the first few bp because the end-repair reaction may introduce a bias towards low methylation.
--three_prime_clip_R1	integer	Instructs Trim Galore to remove spacified number of bp from the 3' end of read 1 (or single-end reads) AFTER adapter/quality trimming has been performed. This may remove some bias from the 3' end that is not directly related to adapter sequence or basecall quality.
--three_prime_clip_R2	integer	Instructs Trim Galore to remove <int> bp from the 3' end of read 2 AFTER adapter/quality trimming has been performed. This may remove some unwanted bias from the 3' end that is not directly related to adapter sequence or basecall quality.
--nextseq	integer	This enables the option '--nextseq-trim=3'CUTOFF' within Cutadapt, which will set a quality cutoff (that is normally given with -q instead), but qualities of G bases are ignored. This trimming is in common for the NextSeq- and NovaSeq-platforms, where basecalls without any signal are called as high-quality G bases. This is mutually exlusive with '-q INT'.
--basename	string	Use specified name (PREFERRED_NAME) as the basename for output files, instead of deriving the filenames from the input files. Single-end data would be called PREFERRED_NAME_trimmed.fq(.gz), or PREFERRED_NAME_val_1.fq(.gz) and PREFERRED_NAME_val_2.fq(.gz) for paired-end data. --basename only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.

Name

Type & Properties

--quality

-q

integer

--phred33

boolean_true

--phred64

boolean_true

--fastqc

boolean_true

--fastqc_args

string

--fastqc_contaminants

file

--fastqc_adapters

file

--fastqc_limits

file

--adapter

-a

string

--adapter2

-a2

string

--illumina

boolean_true

--stranded_illumina

boolean_true

--nextera

boolean_true

--small_rna

boolean_true

--consider_already_trimmed

integer

--max_length

integer

--stringency

integer

--error_rate

-e

double

--gzip

boolean_true

--dont_gzip

boolean_true

--length

integer

--max_n

integer

--trim_n

boolean_true

--no_report_file

boolean_true

--suppress_warn

boolean_true

--clip_R1

integer

--clip_R2

integer

--three_prime_clip_R1

integer

--three_prime_clip_R2

integer

--nextseq

integer

--basename

string

Specific trimming options without adapter/quality trimming

Name	Type & Properties	Description
--hardtrim5	integer	Instead of performing adapter-/quality trimming, this option will simply hard-trim sequences to <int> bp at the 5'-end. Once hard-trimming of files is complete, Trim Galore will exit. Hard-trimmed output files will end in .<int>_5prime.fq(.gz).
--hardtrim3	integer	Instead of performing adapter-/quality trimming, this option will simply hard-trim sequences to <int> bp at the 3'-end. Once hard-trimming of files is complete, Trim Galore will exit. Hard-trimmed output files will end in .<int>_3prime.fq(.gz).
--clock	boolean_true	In this mode, reads are trimmed in a specific way that is currently used for the Mouse Epigenetic Clock.
--polyA	boolean_true	This is a new, still experimental, trimming mode to identify and remove poly-A tails from sequences. When --polyA is selected, Trim Galore attempts to identify from the first supplied sample whether sequences contain more often a stretch of either 'AAAAAAAAAA' or 'TTTTTTTTTT'. This determines if Read 1 of a paired-end end file, or single-end files, are trimmed for PolyA or PolyT. In case of paired-end sequencing, Read2 is trimmed for the complementary base from the start of the reads. The auto-detection uses a default of A{20} for Read1 (3'-end trimming) and T{150} for Read2 (5'-end trimming). These values may be changed manually using the options -a and -a2. In addition to trimming the sequences, white spaces are replaced with _ and it records in the read ID how many bases were trimmed so it can later be used to identify PolyA trimmed sequences. This is currently done by writing tags to both the start ("32:A:") and end ("_PolyA:32") of the reads. The poly-A trimming mode expects that sequences were both adapter and quality before looking for Poly-A tails, and it is the user's responsibility to carry out an initial round of trimming.
--implicon	boolean_true	This is a special mode of operation for paired-end data, such as required for the IMPLICON method, where a UMI sequence is getting transferred from the start of Read 2 to the readID of both reads. Following this, Trim Galore will exit. In it's current implementation, the UMI carrying reads come in the following format Read 1 5' FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF 3' Read 2 3' UUUUUUUUFFFFFFFFFFFFFFFFFFFFFFFFFFFF 5' Where UUUUUUUU is a random 8-mer unique molecular identifier (UMI) and FFFFFFF... is the actual fragment to be sequenced. The UMI of Read 2 (R2) is written into the read ID of both reads and removed from the actual sequence.

Name

Type & Properties

--hardtrim5

integer

--hardtrim3

integer

--clock

boolean_true

--polyA

boolean_true

--implicon

boolean_true

RRBS-specific options

Name	Type & Properties	Description
--rrbs	boolean_true	Specifies that the input file was an MspI digested RRBS sample (recognition site is CCGG). Single-end or Read 1 sequences (paired-end) which were adapter-trimmed will have a further 2 bp removed from their 3' end. Sequences which were merely trimmed because of poor quality will not be shortened further. Read 2 of paired-end libraries will in addition have the first 2 bp removed from the 5' end (by setting '--clip_r2 2'). This is to avoid using artificial methylation calls from the filled-in cytosine positions close to the 3' MspI site in sequenced fragments. This option is not recommended for users of the Tecan Ovation RRBS Methyl-Seq with TrueMethyl oxBS 1-16 kit (see below).
--non_directional	boolean_true	Selecting this option for non-directional RRBS libraries will screen quality-trimmed sequences for 'CAA' or 'CGA' at the start of the read and, if found, removes the first two basepairs. Like with the option '--rrbs' this avoids using cytosine positions that were filled-in during the end-repair step. '--non_directional' requires '--rrbs' to be specified as well. Note that this option does not set '--clip_r2 2' in paired-end mode.
--keep	boolean_true	Keep the quality trimmed intermediate file.

Name

Type & Properties

--rrbs

boolean_true

--non_directional

boolean_true

--keep

boolean_true

Paired-end specific options

Name	Type & Properties	Description
--paired	boolean_true	This option performs length trimming of quality/adapter/RRBS trimmed reads for paired-end files. To pass the validation test, both sequences of a sequence pair are required to have a certain minimum length which is governed by the option --length (see above). If only one read passes this length threshold the other read can be rescued (see option --retain_unpaired). Using this option lets you discard too short read pairs without disturbing the sequence-by-sequence order of FastQ files which is required by many aligners. Trim Galore expects paired-end files to be supplied in a pairwise fashion, e.g. file1_1.fq file1_2.fq SRR2_1.fq.gz SRR2_2.fq.gz ... .
--retain_unpaired	boolean_true	If only one of the two paired-end reads became too short, the longer read will be written to either '.unpaired_1.fq' or '.unpaired_2.fq' output files. The length cutoff for unpaired single-end reads is governed by the parameters -r1/--length_1 and -r2/--length_2.
--length_1 -r1	integer	Unpaired single-end read length cutoff needed for read 1 to be written to '.unpaired_1.fq' output file. These reads may be mapped in single-end mode.
--length_2 -r2	integer	Unpaired single-end read length cutoff needed for read 2 to be written to '.unpaired_2.fq' output file. These reads may be mapped in single-end mode.

Name

Type & Properties

--paired

boolean_true

--retain_unpaired

boolean_true

--length_1

-r1

integer

--length_2

-r2

integer

Output

Name	Type & Properties	Description
--output_dir -o	file required output	If specified all output will be written to this directory instead of the current directory.
--trimmed_r1	file output	Output file for read 1. Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--trimmed_r2	file output	Output file for read 2. Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--trimming_report_r1	file output	Trimming report for read 1. Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--trimming_report_r2	file output	Trimming report for read 1. Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--trimmed_fastqc_html_1	file output	FastQC report for trimmed (single-end) reads (or read 1 for paired-end). Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--trimmed_fastqc_html_2	file output	FastQC report for trimmed reads (read2 for paired-end). Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--trimmed_fastqc_zip_1	file output	FastQC results for trimmed (single-end) reads (or read 1 for paired-end). Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--trimmed_fastqc_zip_2	file output	FastQC results for trimmed reads (read2 for paired-end). Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--unpaired_r1	file output	Output file for unpired read 1. Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.
--unpaired_r2	file output	Output file for unpaired read 2. Only works when 1 file (single-end) or 2 files (paired-end) are specified, but not for longer lists.

Name

Type & Properties

--output_dir

-o

file

required

output

--trimmed_r1

file

output

--trimmed_r2

file

output

--trimming_report_r1

file

output

--trimming_report_r2

file

output

--trimmed_fastqc_html_1

file

output

--trimmed_fastqc_html_2

file

output

--trimmed_fastqc_zip_1

file

output

--trimmed_fastqc_zip_2

file

output

--unpaired_r1

file

output

--unpaired_r2

file

output

Run this component

Run the following command to execute this component with Nextflow:

cat > params.yaml <<'EOM'  
output_dir: "$id.$key.output_dir"  
trimmed_r1: "$id.$key.trimmed_r1.fastq"  
trimmed_r2: "$id.$key.trimmed_r2.fastq"  
trimming_report_r1: "$id.$key.trimming_report_r1.txt"  
trimming_report_r2: "$id.$key.trimming_report_r2.txt"  
trimmed_fastqc_html_1: "$id.$key.trimmed_fastqc_html_1.html"  
trimmed_fastqc_html_2: "$id.$key.trimmed_fastqc_html_2.html"  
trimmed_fastqc_zip_1: "$id.$key.trimmed_fastqc_zip_1.zip"  
trimmed_fastqc_zip_2: "$id.$key.trimmed_fastqc_zip_2.zip"  
unpaired_r1: "$id.$key.unpaired_r1.fastq"  
unpaired_r2: "$id.$key.unpaired_r2.fastq"  
id: "run"  
publish_dir: "output/"  
EOM

nextflow run https://packages.viash-hub.com/vsh/biobox.git \  
  -revision v0.4.0 \  
  -main-script target/nextflow/trimgalore/main.nf \  
  -params-file params.yaml

trimgalore

Input

Trimming options

Specific trimming options without adapter/quality trimming

RRBS-specific options

Paired-end specific options

Output

Run this component

Relationships

Used by

Uses