Hello!

Adapter trimming is necessary for down-stream analysis. This is because: if your insert size is smaller than sequencing cycles, adapter, which is next to the insert, will be sequenced. And this is what your want to removed from your raw data.

Here is my situation, and hope this helps:

1. Sequencing platform: HiSeq 3000, paired-end (2 X 150bp)
2. Kit used for library construction: NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 & 2)

3. Cutadapt was used for adapter trimming, and here is the script: (http://cutadapt.readthedocs.io/en/stable/guide.html#illumina-truseq)

       -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC \ # remove adapter from Read1
       -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT \ # remove adapter from Read2
       -o trimmed.1.fastq.gz -p trimmed.2.fastq.gz \
       reads.1.fastq.gz reads.2.fastq.gz
   

The adapter sequences used in the script above are portions of "index primer" and "universal primer" sequence from NEBNext kit, respectively (as you will find, they are the complementary sequences from 3' to 5'). And this has been nicely addressed by Lesley Sitter.

• NEBNext Universal PCR Primer sequence:
  5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
• NEBNext Index Primers (1-3 for example, indexes are in parentheses):
  Index 1 5'-CAAGCAGAAGACGGCATACGAGAT(CGTGAT)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'
  Index 2 5'-CAAGCAGAAGACGGCATACGAGAT(ACATCG)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'
  Index 3 5'-CAAGCAGAAGACGGCATACGAGAT(GCCTAA)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'

Lastly, here is a figure that might be helpful. As I said in the beginning: if insert size is smaller than sequencing cycles, adapter will be sequenced.

Hi,

I'm not familiar with the NEB kit itself but if found a possible adapter sequence for trimming.

In the NEBkit manual page 5 it states that the adapter is

5'-/5Phos/GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT C/ideoxyU/A CAC TCT TTC CCT ACA CGA CGC TCT TCC GAT C*T-3'

https://www.neb.com/~/media/Catalog/All-Products/33E45F5CD69B497E92C1073F5D551DC7/Datacards%20or%20Manuals/manualE7335.pdf

On this page;

They discuss what type of adapters they use for trimming when using the NEBNext lib prep kit. It seems you also have some sort of index (I'm unfamiliar with the NEBNext protocol, sorry)? In any case these are the adapter sequences they used for trimming;

NEBnext Universal primer
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T

NEBnext Indexed primers 1 - 12 (6-mer indexes)

Index 1 CAAGCAGAAGACGGCATACGAGAT***CGTGAT***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 2 CAAGCAGAAGACGGCATACGAGAT***ACATCG***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 3 CAAGCAGAAGACGGCATACGAGAT***GCCTAA***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 4 CAAGCAGAAGACGGCATACGAGAT***TGGTCA***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 5 CAAGCAGAAGACGGCATACGAGAT***CACTGT***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 6 CAAGCAGAAGACGGCATACGAGAT***ATTGGC***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 7 CAAGCAGAAGACGGCATACGAGAT***GATCTG***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 8 CAAGCAGAAGACGGCATACGAGAT***TCAAGT***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 9 CAAGCAGAAGACGGCATACGAGAT***CTGATC***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 10 CAAGCAGAAGACGGCATACGAGAT***AAGCTA***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 11 CAAGCAGAAGACGGCATACGAGAT***GTAGCC***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T
Index 12 CAAGCAGAAGACGGCATACGAGAT***TACAAG***GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T