Demultiplexing and Trimming Adapters from Reads with q2-cutadapt

:exclamation: :exclamation: :exclamation: NOTE :exclamation: :exclamation: :exclamation:

This tutorial is a work in progress, and is incomplete at the moment. It demonstrates at a high level some of the methods available in the q2-cutadapt plugin available in QIIME 2 2018.2. Please stay tuned here for additional updates as this tutorial is expanded upon in the coming weeks.


Multiplexed reads with the barcodes in the sequence reads can be demultiplexed in QIIME 2 using the q2-cutadapt plugin, which wraps the cutadapt tool. (Multiplexed sequences prepared with the EMP protocol, where barcode reads are in a separate file, as always can be demultiplexed with the q2-demux plugin.) The following tutorial utilizes a toy dataset to illustrate some of the methods in q2-cutadapt.

Download data used in this tutorial

forward.fastq.gz (770 Bytes)
metadata.tsv (53 Bytes)

The data here consists of single-end reads (6 reads total). There are two samples present in the data, with the following barcodes on the 5' end:

Sample    Barcode
------------------
Sample_A  AACCGGTT
Sample_B  CCAAGGTT

Import the multiplexed sequences

$ qiime tools import \
  --type MultiplexedSingleEndBarcodeInSequence \
  --input-path forward.fastq.gz \
  --output-path multiplexed-seqs.qza

Demultiplex the reads

$ qiime cutadapt demux-single \
  --i-seqs multiplexed-seqs.qza \
  --m-barcodes-file metadata.tsv \
  --m-barcodes-column Barcode \
  --p-error-rate 0 \
  --o-per-sample-sequences demultiplexed-seqs.qza \
  --o-untrimmed-sequences untrimmed.qza \
  --verbose

Trim adapters from demultiplexed reads

If there are sequencing adapters or PCR primers in the reads which you'd like to remove, you can do that next as follows.

$ qiime cutadapt trim-single \
  --i-demultiplexed-sequences demultiplexed-seqs.qza \
  --p-front GCTACGGGGGG \
  --p-error-rate 0 \
  --o-trimmed-sequences trimmed-seqs.qza \
  --verbose

Summarize demultiplexed and trimmed reads

$ qiime demux summarize \
  --i-data trimmed-seqs.qza \
  --o-visualization trimmed-seqs.qzv
$ qiime tools view trimmed-seqs.qzv

Regarding paired-end reads

  • The import format for paired-end reads with the barcodes still in the sequence is MultiplexedPairedEndBarcodeInSequence - this format expects two files in a directory (forward.fastq.gz and reverse.fastq.gz).
  • Demultiplexing currently only works if the barcodes are in the forward reads --- we plan to support dual-indexing strategies in a future release of QIIME 2.
  • Demultiplexing is accomplished with the demux-paired command.
  • Filtering/trimming is accomplished with the trim-paired command.
14 Likes

Hi, do you know If in the new version of qiime (2) there is a command that make me paired or do this using the barcode of the forward primer and reverse primer at the same time, in order to differentiate a sample?

Hi @Steph_Hp!

I’m not quite sure what you’re asking for here, but q2-cutadapt supports demultiplexing paired-end reads, and trimming paired-end reads.

This sounds like dual-indexing – please see my note from above:

Hope that answers your questions, if not please let me know! Thanks! :t_rex:

1 Like

An off-topic reply has been split into a new topic: How to demux dual-indexed reads

Please keep replies on-topic in the future.

An off-topic reply has been split into a new topic: Cutadapt trim iontorrent data

Please keep replies on-topic in the future.

An off-topic reply has been split into a new topic: Dual indexing options?

Please keep replies on-topic in the future.

An off-topic reply has been split into a new topic: metadata types specification raises error with cutadapt

Please keep replies on-topic in the future.

A post was split to a new topic: does q2-cutadapt support dual indexed reads?

A post was split to a new topic: importing fastq files and naming conventions

2 posts were split to a new topic: how do I concatenate reads?

A post was split to a new topic: Cutadapt Multicore flag

Hi @thermokarst: is it supported by now if forward and backward sequences have barcodes? If not do you have an estimate when?

Henrik

Support for certain types of dual-indexing strategies was added to q2-cutadapt almost 1 year ago:

2 Likes

A post was split to a new topic: Tutorial for demuxing paired-end reads with barcodes and primers

A post was split to a new topic: No samples found demultiplexing error

A post was split to a new topic: My data is demultiplexed and I just imported them

A post was split to a new topic: Cutadapt demultiplexing with mixed primers

An off-topic reply has been split into a new topic: I am interested in analyzing paired end reads

Please keep replies on-topic in the future.

An off-topic reply has been split into a new topic: help with cutadapt demux-paired

Please keep replies on-topic in the future.