What it does

Trim Galore! is a wrapper script to automate quality and adapter trimming as well as quality control, with some added functionality to remove biased methylation positions for RRBS sequence files (for directional, non-directional (or paired-end) sequencing). It's main features are:

• For adapter trimming, Trim Galore! uses the first 13 bp of Illumina standard adapters ('AGATCGGAAGAGC') by default (suitable for both ends of paired-end libraries), but accepts other adapter sequence, too
• For MspI-digested RRBS libraries, Trim Galore! performs quality and adapter trimming in two subsequent steps. This allows it to remove 2 additional bases that contain a cytosine which was artificially introduced in the end-repair step during the library preparation
• For any kind of FASTQ file other than MspI-digested RRBS, Trim Galore! can perform single-pass adapter and quality trimming
• The Phred quality of basecalls and the stringency for adapter removal can be specified individually
• Trim Galore! can remove sequences if they become too short during the trimming process. For paired-end files Trim Galore! removes entire sequence pairs if one (or both) of the two reads became shorter than the set length cutoff. Reads of a read-pair that are longer than a given threshold but for which the partner read has become too short can optionally be written out to single-end files. This ensures that the information of a read pair is not lost entirely if only one read is of good quality
• Trim Galore! can trim paired-end files by 1 additional bp from the 3' end of all reads to avoid problems with invalid alignments with Bowtie 1

It is developed by Felix Krueger at the Babraham Institute.

Main Settings

• Adapter sequence to be trimmed

  • Automatic detection

    Adapter sequence to be trimmed. Trim Galore will try to auto-detect whether the Illumina universal, Nextera transposase or Illumina small RNA adapter sequence was used.

  • Illumina universal

    Adapter sequence to be trimmed is the first 13bp of the Illumina universal adapter 'AGATCGGAAGAGC' instead of the default auto-detection of adapter sequence.

    
    

    option --illumina

  • Nextera transposase

    Adapter sequence to be trimmed is the first 12bp of the Nextera adapter 'CTGTCTCTTATA' instead of the default auto-detection of adapter sequence.

    
    

    option --nextera

  • Illumina small RNA adapters

    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 -a2 will be set to the Illumina small RNA 5' adapter automatically ('GATCGTCGGACT') unless -a 2 had been defined explicitly.

    
    

    option --small_rna

  • User defined adapter trimming

    Adapter sequence to be trimmed is the sequence entered by the user instead of the default auto-detection of adapter sequence.

    
    

    option -a

• If Single-End Reads

  • Remove <int> bp from the 3' end

    <int> Instructs Trim Galore to remove <int> 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.

    
    

    option --three_prime_clip_R1

• If Paired-End Reads

  • 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 --------------------------->

    R2 <---------------------------

    
    

    or this:

    
    

    R1 ----------------------->

    R2 <-----------------

    
    

    as invalid (whenever a start/end coordinate is contained within the other read).

    
    

    option --t

  • Remove <int> bp from the 3' end of read 1

    <int> Instructs Trim Galore to remove <int> 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.

    
    

    option --three_prime_clip_R1

  • Remove <int> bp from the 3' end of read 2

    <int> 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. Default: OFF.

    
    

    option --three_prime_clip_R2

Advanced Settings

• 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.

  
  

  option -q

• 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 of the 3' end of any read.

  
  

  option -s

• Maximum allowed error rate

  (no. of errors divided by the length of the matching region) (default: 0.1).

  
  

  option -e

• 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 <INT> 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.

  
  

  option --length

• Instructs Trim Galore! to remove INT bp from the 5' end of read 1

  Instructs Trim Galore to remove <INT> 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.

  
  

  option --clip_R1

• Instructs Trim Galore! to remove INT bp from the 5' end of read 2

  Instructs Trim Galore to remove <int> 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.

  
  

  option --clip_R2

• Specify if you would like to retain unpaired reads

  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.

  
  

  option --retained_unpaired

RRBS specific settings

• 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.

  
  

  option -rrbs

• Screen quality-trimmed sequences for 'CAA' or 'CGA' at the start of the read and, if found, removes the first two basepairs

  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.

  
  

  option --non_directional

Trim specific seetings

• Hard-trimming mode

  Instead of performing adapter-/quality trimming, this option will simply hard-trim sequences to N bp at the 5' or 3' -end.

  
  

  options -hardtrim5 and -hardtrim3

• Mouse Epigenetic Clock mode

  In this mode, reads are trimmed in a specific way that is currently used for the Mouse Epigenetic Clock (see here: Multi-tissue DNA methylation age predictor in mouse). Following this, Trim Galore will exit.

  
  

  In it's current implementation, the dual-UMI RRBS reads come in the following format:

  
  

  Read 1 5' UUUUUUUU CAGTA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF TACTG UUUUUUUU 3'

  Read 2 3' UUUUUUUU GTCAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF ATGAC UUUUUUUU 5'

  
  

  Where UUUUUUUU is a random 8-mer unique molecular identifier (UMI), CAGTA is a constant region, and FFFFFFF... is the actual RRBS-Fragment to be sequenced. The UMIs for Read 1 (R1) and Read 2 (R2), as well as the fixed sequences (F1 or F2), are written into the read ID and removed from the actual sequence.

  
  

  option --clock

• PolyA mode

  This is a new, still experimental, trimming mode to identify and remove poly-A tails from sequences. When 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.

  
  

  PLEASE NOTE: The poly-A trimming mode expects that sequences were both adapter and quality trimmed before looking for Poly-A tails, and it is the user's responsibility to carry out an initial round of trimming.

  
  

  option --polyA

• Amplicon mode

  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.

  
  

  option --amplicon