Trimming customed adaptor sequences with trimmomatic

Question

[1215045934]

Hello everyone!

Is it possible to trim the customed adaptors using trimmomatic for exon capture sequencing? If so, how can I create the adaptor fasta file (sequence header etc.)?

The adapters used are below, "BCBCBCBC" stands for the barcodes.

i7: GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-BCBCBCBC-ATCTCGTATGCCGTCTTCTGCTTG

i5: AATGATACGGCGACCACCGAGATCTACAC-BCBCBCBC-ACACTCTTTCCCTACACGACGCTCTTCCGATCT

Thank you in advance!

Answer 1

[GenoMax]

Are you sure these are truly custom adapters? Generally one looks for the core sequence at the beginning of adapter and then once that is found removes everything to the right of that sequence, which will include BCBCBCBC.

You can use bbduk.sh from BBMap suite with whatever adapters you want to use in a multi-fasta file like bbduk.sh in=your.fq out=clean.fq ref=adapter.fa ..... A guide is available.

[1215045934]

Thanks a lot! What is the core sequence you mentioned? I checked the universal adapter sequence file in trimmomatic. I do find the similar sequence to i5 adapter from TruSeq2-PE.fa, Primer1 but not anything similar to i7 adapter. Does that mean trimmomatic will remove the i5 adapter but not i7?

[GenoMax]

Here is an example of TrueSeq adapter sequences (from adapters.fa file in resources directory from BBMap suite)

>TruSeq_Adapter_Index_2
GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG
>TruSeq_Adapter_Index_3
GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG
>TruSeq_Adapter_Index_4
GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG

GATCGGAAGAGCACACGTCTGAACTCCAGTC *ACATCACG* ATCTCGTATGCCGTCTTCTGCTTG
GATCGGAAGAGCACACGTCTGAACTCCAGTC *ACCGATGT* ATCTCGTATGCCGTCTTCTGCTTG
GATCGGAAGAGCACACGTCTGAACTCCAGTC *ACTTAGGC* ATCTCGTATGCCGTCTTCTGCTTG
GATCGGAAGAGCACACGTCTGAACTCCAGTC *ACTGACCA* ATCTCGTATGCCGTCTTCTGCTTG

So you can see that the differences are the indexes (or barcodes, I have separated them out and added a * to delimit) while the core sequences on either side are identical and match the i7 sequence you have.

[1215045934]

Thank you so much! Is it possible to trim them in trimmomatic? It seems that the i7 sequence is not in the build-in adapter sequence in trimmomatic

[GenoMax]

Check other TruSeq*.fa files in Trimmomatic.

[1215045934]

Yes I did check all the TruSeq-*-PE files in Trimmomatic but I did not find the i7 sequence.

[GenoMax]

You could use adapters.fa file from BBMap suite or simply use bbduk.sh :-)

[1215045934]

Thank you so much! I will try it!

[1215045934]

I did QC after trimming with both trimmomatic and bbduk.sh but there is still some Illumnina Universal Adapter left. Would you mind giving me some suggestions?

[GenoMax]

What is the exact command you used with bbduk.sh?