I am currently working on a project where I need to trim the adapters off of some single end read RNA-Seq data, and I want to know which sequences to cleave. Illumina TruSeq adapters were used. I have tried following Tuft's explanation and make sense of Illumina's video, but I am left with several unresolved questions.
The TruSeq Universal adapter, seems to be what binds to the flow cell and is
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
The TruSeq Adapter Index N is
GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-NNNNNN-ATCTCGTATGCCGTCTTCTGCTTG
My impression of how the cDNA fragment attached to the adapters is :
(flow cell)
||
||
|| (Index Adapter) (Universal Adapter)
|| 3' 5' 3' 5'
|| GTTCGTCTTCTGCCGTATGCTCTA-NNNNNN-CACTGACCTCAAGTCTGCACACGAGAAGGCTAGA TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA
|| |||||||||||||| |||||||||||||| <----- Must get denatured!
|| 5' AATGATCGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT--(5' MY DNA FRAGMENT 3')--AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC‐NNNNNN‐ATCTCGTATGCCGTCTTCTGCTTG
||-------------TTACTAGCCGCTGGTGGCT 3' 5' 3'
||
|| ^(Flow cell oligo) ^(Universal Adapter) ^(Index Adapter)
||
|| TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGTG
||
|| ^(Read 1 Primer) ^(Read 2 Primer)
||
||
|| TAGAGCATACGGCAGAAGACGAAC----------||
||
|| ^(Flow cell oligo)
||
Here are my questions :
Is this image correct in the single stranded case?
Do I have the correct primer binding site (used for the actual sequencing)?
Do I have the correct oligo for paired end reads, i.e.
TAGAGCATACGGCAGAAGACGAAC---------?Do I have the correct primer (and it's site) for the index read?
Per Illumina, I guess I need to include this : "Oligonucleotide sequences © 2007-2013 Illumina, Inc. All rights reserved. Derivative works created by Illumina customers are authorized for use with Illumina instruments and products only. All other uses are strictly prohibited."
