Demultiplexing Help

Hi,

I am fairly new to QIIME2 and I am trying to run some data. I extracted barcodes and reads (renamed to sequences) through QIIME1 and now I am trying to import and demultiplex which I believe is the source of my problem. It has no problems running through the import (type EMPSingleEndSequences) and demultiplexing commands, however the .qzv file I get from the summarization only has three of 84 samples.

After running

qiime dada2 denoise-single
--i-demultiplexed-seqs demultiplexed-seqs.qza
--p-trim-left 8
--p-trunc-len 0
--o-representative-sequences rep-seqs-dada2.qza
--o-table table-dada2.qza

I get this error:
No reads passed the filter. trunc_len (0) may be longer than read lengths, or other arguments
(such as max_ee or trunc_q) may be preventing reads from passing the filter.

Is there anything wrong with what I am doing?

Thanks!

Hey @Dean - I suspect something is quite right with your barcodes. Can you provide the following:

  • What version of QIIME 2 are you running?
  • Can you confirm that your reads are single-end? I see you are running dada2 denoise-single, but want to be sure.

I was going to ask you to run head on all of your files (before and after extracting barcodes in Q1), but, I realized this might be a great use case for q2-cutadapt! You should be able to skip Q1 here, and just import your raw (barcoded) reads, then use the cutadapt demux-single command to demultiplex these reads. How about you give that a shot (check out the Community Tutorial for some assistance, too)? That might be easier, and we can’t really provide support for Q1 anymore, which is likely where the issue you originally reported is cropping up. Keep us posted! :t_rex:

Hi,

  • I am currently running QIIME 2 2017.12
  • The reads are single-end, multiplexed.

I was given a .qual and .fasta file which I converted into a .fastq file and extracted the barcodes and reads. I have just tried using q2-cutadapt (following the community tutorial) and have encountered this error:

q2-CasavaOneEightSingleLanePerSampleDirFmt-20xn2609/[SampleID].VSL_[Barcode]_L001_R1_001.fastq.gz is not a(n) FastqGzFormat file:
Missing sequence for record beginning on line 13

when I tried using the reads.fastq.gz (and barcodes.fastq.gz in case that worked), running cutadapt demux-single. Both times I tried running it, they returned a different SampleID and sequence for record line error.

Is there a different way to extract reads from the .qual/.fasta files other than going through Q1?

Thanks for helping out!

Hi @Dean!

If you are going to demultiplex using the EMP protocol (demux emp-single), this is fine, but it won’t work if you’re planning on using q2-cutadapt's demultiplexing methods.

Have you had a chance to familiarize yourself with the Importing tutorial? There are some descriptions in that tutorial that are relevant to what you are working on, and ultimately these errors come down to some fundamental mismatches between data and the commands you are trying to run on it.

Okay, so moving forward, I would recommend the following. Convert your fast+qual to fastq using whatever tool you did to accomplish that previously. Import that one fastq file as MultiplexedSingleEndBarcodeInSequence, then follow the q2-cutadapt community tutorial to demultiplex these data. :crossed_fingers:

No problem, that is what we are here for! Thanks for working with us to make QIIME 2 a great place to be! :t_rex: :balloon:

Hi,

I still seem to be getting the same error following what was said here:

Okay, so moving forward, I would recommend the following. Convert your fast+qual to fastq using whatever tool you did to accomplish that previously. Import that one fastq file as MultiplexedSingleEndBarcodeInSequence, then follow the q2-cutadapt community tutorial to demultiplex these data. :crossed_fingers:

I followed through restarting at step one. This is what I did:

  1. Convert fasta+qual to fastq
  2. Converted fastq to fastq.gz
  3. Imported as MultiplexedSingleEndBarcodeInSequence
  4. Followed the q2-cutadapt community tutorial (even tried changing error-rate to 0.1)

The error I received every time is this (SampleID and BarcodeSequence is there for confidentiality):

Plugin error from cutadapt:

/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-0w03hmww/[SampleID].VSL_[BarcodeSequence]_L001_R1_001.fastq.gz is not a(n) FastqGzFormat file:

Missing sequence for record beginning on line 9

I have tried checking the validity of the archive (using the gzip -tv command) as well as checking if the multiplexed-seqs.qza was valid (used qiime tools validate). Both returned okay and valid.

Could there something wrong with the way I imported file or would it be a problem with my mapping file?

Hey @Dean! We have seen a few reports like this in the last couple of days, for example:

This thread can fill you in on the details. We are working on the issue right now and will keep you posted. Thanks! :t_rex:

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