Adapters list received for PE Illumina RNA-seq

[Ohad]

Hi,

I would appreciate some help.
We are analyzing PE RNA-seq results done with Illumina.

I have received a list of 11 adapters which were used in the process but I cant figure out how exactly.
I have spend a lot of time figuring out the Adapter-illustration.pdf file from this web and it helped me identify first adapter on the list as one of the PE primer used for PCR in the library stage.

But what are the others ? and why do they vary in 6 bases around the middle ?

Here is the list:

1 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT


2 GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG
3 GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG
4 GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG
5 GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG
6 GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG
7 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG
8 GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG
9 GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG
0 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG
1 GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG

[mastal]

The 6 bases in the middle are the index/barcode for multiplexed samples.

How many samples are you analysing? If your samples were multiplexed they should have the barcode or sample number in the fastq header of the reads.

[Ohad]

Thank you mastal
You are right, it seems this adapters list relates to 10 sample.
First one for all, and then the following 10 hold different indexes.

Thanks again for your quick reply

[Ohad]

I read more about multiplexed sequencing with PE

So, for adapters trimming I assume I need to trim Adapter #1 as is ,and one of the other adapters (depends on the file im working on) when it's complementary and reverese ?

[mastal]

Just to double check, have a look at what adapter sequences are actually present in your reads, using 'grep'.

[fkrueger]

Hi Ohad,

Both adapters on either side share the first 13bp AGATCGGAAGAGC (the sequences you linked are missing the first A from the A-tailing procedure), so it should be sufficient to just use this sequence for trimming (Trim Galore uses this sequence by default). This should trim both read 1 and read 2 of your files, independently of the index used (don't forget to use --paired).

Best, Felix

[Ohad]

Thank you felix.

I assume you suggest that since TG will cut all remaining bases downstream to the 13bp adapter matched anyhow (if they ever exist on a particular read, and probably containing remaining adapter sequence).

Am I right ?

[mastal]

Yes, that's right.

[Ohad]

Thank you both for your wise suggestions.