Qiime 1.9.1: dual index: illumina: Produces no output in split library fastq

[Sanjeev Sariya]

Hi There,

I've V3-V4 region sequenced using fadrosh et al

I'm coming from the thread

Both Forward and reverse barcodes are of length 12

Forward primer is of length 16

Reverse Primer is of length 21 

I'm using QIIME - 1.9.1

Here are the steps followed:

1- I used fastq-join (ea-utils) with an overlap of 30 to join the reads

It generated 3 files: 

fastqjoin.join.fastq, fastqjoin.un1.fastq, and fastqjoin.un2.fastq 

2- Next, I extracted barcode

I use fastqjoin.join.fastq from previous step:

Command:

extract_barcodes.py -f fastqjoin.join.fastq -c barcode_paired_stitched --bc1_len 12 --bc2_len 12

It generates 2 outputs:

reads.fastq and  barcodes.fastq

3- Next, I'm to use split_libraries_fastq

I use  barcodes.fastq from step 2, and fastqjoin.join.fastq from step 1.

Command:

split_libraries_fastq.py -i fastqjoin.join.fastq -m qiime_mapping.txt -q 19 -o slout -b barcodes.fastq --barcode_type 12

Unfortunately, I don't get any sequences.

My run ends with an exception raised for 

"Mean of empty slice"

--

I've following queries to make sure I'm not barking at the wrong tree:

1) Am I using correct set of file at step 3?

2) In my mapping file, what barcodes should I be using? 

a) plainly concatenate forward and reverse? (without any rev com of reverse barcode)

I got this information from this

b) Or, concatenate forward primer and reverse the barcode for reverse primer?

c) Something I do not know of.

 

3)

In my mapping file I've column for forward and reverse primer. 

Attached mapping file for reference.

Am I doing anything wrong in it? I validated it using validate script from qiime. No errors were found.

Thank you very much for your patience.

[Sanjeev Sariya]

Read length 256. 

[zech xu]

You commands seem legitimate to me. could you send the whole message of exception? one possibility is that your barcode is revers (complementary) to what you provided in your mapping file. have you checked that?

[Sanjeev Sariya]

Hi Zech,

Thanks for your reply, and apologies for getting back late. I gave up on this data. Shall connect if this kind of data flows in more. 

Appreciate your reply, and support!