Demultiplexing fastq file with samples that have an identical barcode tag.

Question

[m.niwano]

Hello I am currently working on the reanalysis of a published study that investigated eukaryotic community during seasonal algal blooms.

To reanalyse the dataset, I downloaded a single multiplexed fastq file (454 pyrosequencing) from the NCBI, which contains sequences from the samples over the course of 3 years in the Eastern English Channel.

As I am demultiplexing the file into separate fastq files, I can not separate some of the samples because some samples have the identical barcode tag for the sequencing (e.g.Barcode tag for samples collected on 26/02/2013 and 21/06/2012 is TAGTATCAGC).

Which tool or a way can I use to distinguish two separate samples from an identical barcode tag?

Here is the barcode tags listed on the NCBI https://www.ncbi.nlm.nih.gov/sra/SRX768577

Thank you

[GenoMax]

I have not worked with 454 data for a while but in general you can't separate samples if they have an identical index. Perhaps there was a typo/submission error in SRA record. Have you checked the publication to see if it contains correct indexes (is there a paper)?

[m.niwano]

Thanks for your advice, but I do not think it was a type or mistake in the SRA record since there are more than 5 barcode seuqneces that are identical between two different samples. (see below)

 ACATACGCGT 27-09-11-EEC-Coast_L001_R1.fastq
 ACATACGCGT 07-02-12-EEC-Coast_L001_R1.fastq
 ACGCGAGTAT 07-03-11-EEC-Coast_L001_R1.fastq
 ACGCGAGTAT 27-05-13-EEC-Offshore_L001_R1.fastq
 ACGCTCGACA 05-06-12-EEC-Coast_L001_R1.fastq
 ACGCTCGACA 24-04-13-EEC-Coast_L001_R1.fastq

https://doi.org/10.1093/femsec/fiv034 This is the paper that used this datasets to analyze the marine diversity in the EEC, but it doesn't not mention regarding the index they used.