USAGE: trim_galore [options] -h/--help Print this help message and exits. -v/--version Print the version information and exits. -q/--quality Trim low-quality ends 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). Default Phred score: 20. --phred33 Instructs Cutadapt to use ASCII+33 quality scores as Phred scores (Sanger/Illumina 1.9+ encoding) for quality trimming. Default: ON. --phred64 Instructs Cutadapt to use ASCII+64 quality scores as Phred scores (Illumina 1.5 encoding) for quality trimming. --fastqc Run FastQC in the default mode on the FastQ file once trimming is complete. --fastqc_args "" Passes extra arguments to FastQC. If more than one argument is to be passed to FastQC they must be in the form "arg1 arg2 etc.". An example would be: --fastqc_args "--nogroup --outdir /home/". Passing extra arguments will automatically invoke FastQC, so --fastqc does not have to be specified separately. -a/--adapter 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. Also see '--illumina', '--nextera' and '--small_rna'. If no adapter can be detected within the first 1 million sequences of the first file specified Trim Galore defaults to '--illumina'. -a2/--adapter2 Optional adapter sequence to be trimmed off read 2 of paired-end files. This option requires '--paired' to be specified as well. --illumina Adapter sequence to be trimmed is the first 13bp of the Illumina universal adapter 'AGATCGGAAGAGC' instead of the default auto-detection of adapter sequence. --nextera 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 Adapter sequence to be trimmed is the first 12bp of the Illumina Small RNA Adapter 'ATGGAATTCTCG' instead of the default auto-detection of adapter sequence. --stringency 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. -e Maximum allowed error rate (no. of errors divided by the length of the matching region) (default: 0.1) --gzip 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 Output files won't be compressed with GZIP. This option overrides --gzip. --length Discard reads that became shorter than length INT because of either quality or adapter trimming. A value of '0' effectively disables this behaviour. Default: 20 bp. For paired-end files, both reads of a read-pair need to be longer than bp to be printed out to validated paired-end files (see option --paired). If only one read became too short there is the possibility of keeping such unpaired single-end reads (see --retain_unpaired). Default pair-cutoff: 20 bp. -o/--output_dir If specified all output will be written to this directory instead of the current directory. --no_report_file If specified no report file will be generated. --suppress_warn If specified any output to STDOUT or STDERR will be suppressed. --clip_R1 Instructs Trim Galore to remove 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. Default: OFF. --clip_R2 Instructs Trim Galore to remove 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. Please refer to the M-bias plot section in the Bismark User Guide for some examples. Default: OFF. --three_prime_clip_R1 Instructs Trim Galore to remove bp from the 3' end of read 1 (or single-end reads) 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. Default: OFF. --three_prime_clip_R2 Instructs Trim Galore to remove 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. Default: OFF. --path_to_cutadapt You may use this option to specify a path to the Cutadapt executable, e.g. /my/home/cutadapt-1.7.1/bin/cutadapt. Else it is assumed that Cutadapt is in the PATH. RRBS-specific options (MspI digested material): --rrbs Specifies that the input file was an MspI digested RRBS sample (recognition site: CCGG). Sequences which were adapter-trimmed will have a further 2 bp removed from their 3' end. This is to avoid that the filled-in C close to the second MspI site in a sequence is used for methylation calls. Sequences which were merely trimmed because of poor quality will not be shortened further. --non_directional 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. --keep Keep the quality trimmed intermediate file. Default: off, which means the temporary file is being deleted after adapter trimming. Only has an effect for RRBS samples since other FastQ files are not trimmed for poor qualities separately. Note for RRBS using MseI: If your DNA material was digested with MseI (recognition motif: TTAA) instead of MspI it is NOT necessary to specify --rrbs or --non_directional since virtually all reads should start with the sequence 'TAA', and this holds true for both directional and non-directional libraries. As the end-repair of 'TAA' restricted sites does not involve any cytosines it does not need to be treated especially. Instead, simply run Trim Galore! in the standard (i.e. non-RRBS) mode. Paired-end specific options: --paired 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 ... . -t/--trim1 Trims 1 bp off every read from its 3' end. This may be needed for FastQ files that are to be aligned as paired-end data with Bowtie. This is because Bowtie (1) regards alignments like this: R1 ---------------------------> or this: -----------------------> R1 R2 <--------------------------- <----------------- R2 as invalid (whenever a start/end coordinate is contained within the other read). --retain_unpaired 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. Default: OFF. -r1/--length_1 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. Default: 35 bp. -r2/--length_2 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. Default: 35 bp. Last modified on 06 May 2015.