MiSeq seq of samples prepared using Illumina's Nextera protocol

[richard rodrigues]

Hi,

I have data from the paired-end MiSeq of 16S rRNA prepared using http://supportres.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf

If I understand it correctly, the general layout is:
P5 - Index2 - fwd overhang_adapter-fwd_primer - DNA - rev_primer-rev overhang_adapter - Index1 - P7

The sequencing facility have removed the barcodes and provided us demultiplexed files, i.e. I have two fastq file per sample (S1_read1.fastq and S1_read2.fastq). I have to remove the adapters and primers and then join them to form one read.

So I am trying to do the following:
1. Make the adapters.fasta file with fwd overhang_adapter-fwd_primer as one adapter and the rev overhang_adapter-rev_primer as second adapter.
2. I want to delete the:
   (i) fwd overhang_adapter-fwd_primer from beginning of read 1
   (ii) rev_primer-rev overhang adapter from end of read 1
   (iii) rev overhang_adapter-rev_primer from beginning of read 2
   (iv) reverse complement of fwd overhang_adapter-fwd_primer from end of read 2

In the help documentation, I came across the following which is very confusing to me, "Adapter sequences with _5p in their label will match 'end's, and sequences with _3p in their label will match 'start's, otherwise the 'end' is auto-determined."

• Does this mean I do the following in adapters.fasta: label_5p for rev_primer-rev overhang adapter and label_3p for fwd overhang_adapter-fwd_primer or am I misunderstanding something?
• I am guessing that the tool can remove (i) and (iii), how can I get it to remove (ii) and (iv)?
• For my particular case, if you think ea utils might not be suitable, do you know if cutadapt or pandaseq could be easy to use?

Thanks very much in advance for your time and help.

-Rich

[richard rodrigues]

I think (ii) should be reverse complement of rev overhang_adapter-rev_primer from end of read 1