how to split barcoded files from master files to load into Qiime

Technical Support
1

Dear All,

I am experiencing difficulties importing my libraries into QIIME. Each sample's contains both forward and reverse barcodes. Prior to import, demultiplexing raw data with my Linux command line generates FASTA files, which are not supported. Please assist me.
Thank you.

Regards
Divya

2

Hi @divya_ch,
Welcome in the forum!

What tool do you use to demultiplex the raw data? I believe there should be an option to produce demultiplexed fastq files.

However as alternative, you could import the raw data and demultiplex within the qiime pipleine using this plug in:
https://docs.qiime2.org/2022.11/plugins/available/cutadapt/demux-paired/

Please see cutadapt manual for details on how to use to demultiplex. However, in the context of qiime2, I believe it requires the same barcode in bot forward and reverse

The following guide could also be helpful to see what are the available options to imort and demultiplex the data!
https://forum.qiime2.org/t/importing-and-demultiplexing-guide/13618

Cheers
Luca

3

Hi Luca,
Thanks for your reply.

i use following commands demultiplex my samples

grep CTCC[A,C,T,G][A,C,T,G][A,C,T,G]GTGCCAGC[A,C]GCCGCGGTAA[A,C,T,G]*ATTAGA[A,T]ACCC[C,G,T][A,G,T]GTAGTCC[A,C,T,G][A,C,T,G][A,C,T,G]CCTC inputfile > Sample#001 ;
grep TGATT[A,C,T,G][A,C,T,G][A,C,T,G]GTGCCAGC[A,C]GCCGCGGTAA[A,C,T,G]*ATTAGA[A,T]ACCC[C,G,T][A,G,T]GTAGTCC[A,C,T,G][A,C,T,G][A,C,T,G]CCTC inputfile > Sample#002 ;
grep CGGATT[A,C,T,G][A,C,T,G][A,C,T,G]GTGCCAGC[A,C]GCCGCGGTAA[A,C,T,G]*ATTAGA[A,T]ACCC[C,G,T][A,G,T]GTAGTCC[A,C,T,G][A,C,T,G][A,C,T,G]CCTC inputfile > Sample#003

how to use the same barcodes to demultiplex in QIIME2?

6

Hi @divya_ch,

what file do you have as raw data?
fastq files? Sequence files form the sequencer?

Cheers
Luca

7

Dear luca,

Fastq files containing pooled amplicon sequnces. Eash sample consists of dual barcodes for both forward and reverse primers.

Regards
Divya

10

Dear @divya_ch
first just a note, while answering here please tag me with '@' sign in fron of name, so I will receive
a notification!

Is the same barcode present in both forward and reverse read? If so, you can use q2-cutadapt to demultiplexd them. Else, you need to demultiplex them using cutadapt outside qiime2, then import the demultiplexed file using the manifest import method.
Cutadapt is already installed in your qiime2 environment, but the options you need are not available via the q2-cutadapt plug in, so please follow instructions at:
https://cutadapt.readthedocs.io/en/stable/guide.html#combinatorial-demultiplexing

Hope it helps
Luca

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12

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