Read trimming with =trimmomatic

Download and Install

Download the "binary" version of trimmomatic from the Usadel lab.

It's helpful to a create a shortcut so that you can call the command by typing trimmomatic rather than remembering the more complicated java command line every time. You can do this by adding a line like this to your bash profile:

alias trimmomatic='java -jar /Users/name/local/bin/trimmomatic-0.XX.jar'

Replace the path with the path to the *.jar file and the XX with the version number.

The command line does not provide very detailed help, so keep the Manual (PDF) handy!

Notes on Choosing or Creating an Adaptor File

One of the main advantages of using trimmomatic is that is deals well with collapsing and trimming paired-end sequencing of fragments that are shorter than the read length, where the read extends into the adaptors at the end of each read.

In this PE mode, trimmomatic will collapse these sequences to a single (and now unpaired) read, so that it will not be counted as sequencing of two independent DNA molecules This is important when analyzing sequences isolated from a mixed population.

The manual didn't exactly make it clear what adaptor sequences need to be included to get this trimming to work properly. So let's assume you have constructed a library with this format.

Adaptor1-Forward‑Sequenced-DNA‑Adaptor2-Forward
Adaptor1-Complement‑Read_R1‑Adaptor2-Complement

If sequencing reads through the insert into the adaptor, then your paired reads (R1 and R2) will look like this:

R1: Sequenced-DNA‑Adaptor2-Forward
R2: Adaptor2(Reverse Complement) - Sequenced-DNA (Reverse Complement)

In this case, your adaptor file needs to include the lines:

>Prefix_Name/1

Adaptor1-Forward
>Prefix_Name/2

Adaptor2-Forward

Note: It is OK to put ambiguous bases (N) in the adaptor sequences if you have a bar code in your adaptors.

Useing with =gdtools

If you are using gdtools, the command to generate your trimming commands. This will re-combine all of the R1 and R2 sequences into two files that will no longer have the same number of sequences in them (so they are not suitable for input for paired-end mapping). You can use the -p option to preserve the pairing in the output files (creating the normal 4 rather than 2 output files).