Orientation of mate-pairs for paired end illumina reads

Commands in mothur
1

Hello,
I have a MiSeq run which produced two fastq files; one of which contained all forward reads, and the second all reads in the same orientation that needed to be reverse complimented.

In my data each fastq file shows a mixture of orientated reads:

Fastq1
BARCODE–F. PRIMER–NNNN
BARCODE–R.PRIMER–NNNN

Fastq 2
BARCODE–R.PRIMER–NNNN
BARCODE–F. PRIMER–NNNN

I guess that make_contigs script designed to join overlapping PE reads will not be able to take into consideration the mixture of Fwd and Rev sequences in each file?
is this normal, or should I be seeing all Fwd and Rev primer sequences in separate Fastqs?
is there any way to analyses such data by MOTHUR?
I also have barcode with variable length?
I have used mothur for 454 data analysis but i a new to Illumina data and not good in custom scripting.
Any help for demultiplexing of such a complex data would be really great.
Cheers,
Sunil Mundra

2

Hello,
Looking forward to have feedback here, than i can proceed further with my analysis
Regards
Sunil :slight_smile:

3

I will add the reorient parameter to the make.contigs command for version 1.34.0 to help with this situation.

4

Hi,That would be great.
How i should proceed for now, in this situation?
Is this something wrong in sequencing or it is usual for illumina sequencing?
Also while using make.contigs, we are not able to do quality filtering of the sequencing as we didnt get any quality file along with fasta file?
Regards

Sunil

5

Hi,
Is this mothur version 1.34.0 with re-orient parameter has been release, i am unable to download it?
How we can do quality filtering for paired end illumina reads in mothur?
regards
sunil