Barcode in the name of the sequence (fastq Illumina)

[Touchpa]

Hi,

Sorry I am a beginner in QIIME, I just have few questions.

My data are formatted like :

@EAS139:136:FC706VJ:2:5:1000:12850 1:Y:18:ATCACG

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

+

BBBBCCCC?<A?BC?7@@???????DBBA@@@@A@@

So the barcode is in the name of the sequence. My sequence is kind of demultiplexed. But it's my raw data.
I have a lot of sample and I planed to use QIIME for analyse.

Do I have to use split_libraries_fastq.py ? because it needs the .map file.
The .map file can recognize the barcode in the name of the sequence or I have to copy the barcode in the front of each sequences ?

 Thanks.

[Tony Walters]

Hello Touchpa,

You will still want to create the separate barcode fastq file for input (-b) with split_libraries_fastq.py 

This should be possible with the extract_barcodes.py script (see http://qiime.org/scripts/extract_barcodes.html, last example covers barcodes in labels). So in your case, the parameters should be -c barcode_in_label --bc1_len 6 --char_delineator ':'

-Tony 

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[Touchpa]

Hi TonyWalters  and thanks for your reply.

So the command you gave me produced a barcode.fastq file

Then I made a map.txt file with :

#SampleID    BarcodeSequence    LinkerPrimerSequence
id1    ATCACG

Then I run:

split_libraries_fastq.py -o slout/ -i mydata.fastq -b processed_seqs/barcodes.fastq -m map.txt --barcode_type 6

But I got an error:

RuntimeWarning: Mean of empty slice.
  warnings.warn("Mean of empty slice.", RuntimeWarning)  

Am I doing wrong ?

[Tony Walters]

Do you only have a single sample? Called id1?

You want to run validate_mapping_file.py after you've made sure the mapping file is complete (http://qiime.org/documentation/file_formats.html#metadata-mapping-files).

You might also visually inspect your input files for any empty sequences (were all of the input sequences for extract_barcodes.py all formatted in the same way, with a 6 base pair barcode at the end following the last ":" character):

processed_seqs/barcodes.fastq
mydata.fastq

[Touchpa]

I ve run validate_mapping_file.py and no message error.
And yes I just have 1 sample.

Can I use this command instead of running extract_barcodes.py   ?

split_libraries_fastq.py -i mydata.fastq --sample_id id1 -o slout/ --barcode_type 'not-barcoded'

what is the advantage ?

[Tony Walters]

Yes, you can run split_libraries_fastq.py with that approach. As you are only expecting a single sample, this is the quickest way to demultiplex the data.

[Touchpa]

If I have a lot of samples. Which way is better ?

[Tony Walters]

If that is the case, then you want to get a list of the barcodes and the associated samples, and create a complete metadata mapping file using the format previously linked.

You will need to figure out if all of your reads have the barcode in the label, and if the scheme for the barcode is consistent-is it always at the end of the label? Is it always the same length? Are there any reads that are missing the barcode?

The format where the barcodes are in the label as you have them are almost certainly from some sort of parsing at your sequencing center, so they may have answers to these questions as well.

-Tony