*Trimmomatic*

A flexible read trimming tool for Illumina NGS data

Trimmomatic performs a variety of useful trimming tasks for illumina paired-end and single ended data.The selection of trimming steps and their associated parameters are supplied on the command line.

The current trimming steps are:

ILLUMINACLIP: Cut adapter and other illumina-specific sequences from the read.

SLIDINGWINDOW: Perform a sliding window trimming, cutting once the average quality within the window falls below a threshold.

LEADING: Cut bases off the start of a read, if below a threshold quality

TRAILING: Cut bases off the end of a read, if below a threshold quality

CROP: Cut the read to a specified length

HEADCROP: Cut the specified number of bases from the start of the read

MINLEN: Drop the read if it is below a specified length

The Adapter Fasta

Illumina adapter and other technical sequences are copyrighted by Illumina, which makes it illegal for us to make them available without a licence. As such, you need to get access to the illumina sequences from another source, and create an appropriate adapter FASTA.

To make this fasta, you must first understand how it will be used. Trimmomatic uses two strategies for adapter trimming: Palindrome and Simple

With 'simple' trimming, each adapter sequence is tested against the reads, and if a sufficiently accurate match is detected, the read is clipped appropriately.

'Palindrome' trimming is specifically designed for the case of 'reading through' a short fragment into the adapter sequence on the other end. In this approach, the appropriate adapter sequences are 'in silico ligated' onto the start of the reads, and the combined adapter+read sequences, forward and reverse are aligned. If they align in a manner which indicates 'read-through', the forward read is clipped and the reverse read dropped (since it contains no new data).

Naming of the sequences indicates how they should be used. For 'Palindrome' clipping, the sequence names should both start with 'Prefix', and end in '/1' for the forward adapter and '/2' for the reverse adapter.All other sequences are checked using 'simple' mode. Sequences with names ending in '/1' or '/2' will be checked only against the forward or reverse read. All other sequences will be checked against both the forward and reverse read. If you want to check for the reverse-complement of a specific sequence, you need to specifically include the reverse-complemented form of the sequence as well, with another name.

The thresholds used are a simplified log-likelihood approach. Each matching base adds just over 0.6, while each mismatch reduces the alignment score by Q/10. Therefore, a perfect match of a 20 base sequence will score just over 12, while 25 bases are needed to score 15. As such we recommend values between 12 - 15 for this parameter. For palindromic matches, the entire read sequence plus (partial) adapter sequences can be used - therefore this threshold can be higher, in the range of 30-40. The 'seed mismatch' parameter is used to make alignments more efficient, specifying the maximum base mismatch count in the 'seed' (16 bases). Typical values here are 1 or 2.

Citation

Lohse M, Bolger AM, Nagel A, Fernie AR, Lunn JE, Stitt M, Usadel B. RobiNA: a user-friendly, integrated software solution for RNA-Seq-based transcriptomics. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W622-7.

Contacts

Anthony Bolger bolger at mpimp-golm dot mpg dot de

Federico M. Giorgi federico dot giorgi at gmail dot com

Wrapped for Galaxy by:

Simon Gladman (VBC/VLSCI) simon dot gladman at monash dot edu