Problem for demultiplexing Miseq Paired end reads using make.contigs with oligos file

Commands in mothur
1

I am having trouble to demultiplex and trim primers and barcodes from a fastq paired-end file.

I have one single pair of fastq file from a paired-end MiSeq run and an oligo file like this:

primer	AYTGGGYDTAAAGNG	CCGTCAATTYHTTTRAGT	V4
primer	CCGTCAATTYHTTTRAGT	AYTGGGYDTAAAGNG	V5
BARCODE	ACCTGATCCGCA	ACCTGATCCGCA	NOD_1
BARCODE	GTTGACCATCGC	GTTGACCATCGC	NOD_2
BARCODE	TGACTGCGTTAG	TGACTGCGTTAG	NOD_3

I used this command:
make.contigs(file=stability.files, oligos=pool94.oligos,bdiffs=1, processors=8)

However, I get 99.99% of the reads on the scrap file without trimming or any information of each detected sample.
My groups file looks like:

M00430_5358_000000000-CCGRN_1_1101_10216_15499	pool94
M00430_5358_000000000-CCGRN_1_1101_10594_20693	pool94
M00430_5358_000000000-CCGRN_1_1101_10676_10175	pool94
M00430_5358_000000000-CCGRN_1_1101_10878_13512	pool94
M00430_5358_000000000-CCGRN_1_1101_11087_21332	pool94
M00430_5358_000000000-CCGRN_1_1101_12496_11697	pool94
M00430_5358_000000000-CCGRN_1_1101_12859_24247	pool94

My question is, can mothur detect the barcode and primers anywhere on the read? or is it expecting it to be at the beginning of the read?

My Reads look like this:
The first match is the bc and the second the primer:

Thanks for your attention.

Can someone clarify it to me?

2

Hi Tiago,

Yes, the sequences are expected to start with the barcode sequence. If there are a fixed number of bases (or sequence) that precede the barcode for each sequence you could concatenate N’s or the expected sequences to the barcode.

Pat

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Hi pschloss,

My Problem was that, the number of reads preceding the BC is variable. I had to use a third party software to demultiplex those samples. But Thanks for the clarification.

Tiago

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