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Alternative methods of read-joining in QIIME 2

Note

This tutorial does not cover read-joining and denoising with DADA2. Instead, this tutorial focuses on alternative approaches to analyzing paired-end reads in QIIME 2. If you are interested in joining and denoising reads with DADA2, the Atacama soil microbiome tutorial illustrates how to use qiime dada2 denoise-paired for this purpose. If you plan to use DADA2 to join and denoise your paired end data, do not join your reads prior to denoising with DADA2; DADA2 expects reads that have not yet been joined, and will join the reads for you during the denoising process.

In QIIME 2, we use the term single-end reads to refer to forward or reverse reads in isolation; we use the term paired-end reads to refer to forward and reverse reads that have not yet been joined; and we use the term joined reads to refer to forward and reverse reads that have already been joined (or merged). It is important to understand which of these terms apply to your data, as this will determine what steps are necessary to analyze your paired-end data.

It is currently possible to join paired-end reads in QIIME 2 using the q2-vsearch plugin, or to import reads that have been joined outside of QIIME 2 (for example, with fastq-join, see Importing pre-joined reads for more details). This tutorial will cover both of these processes.

Obtain the data

Start by creating a directory to work in.

mkdir qiime2-read-joining-tutorial
cd qiime2-read-joining-tutorial

Next, download the following SampleData[PairedEndSequencesWithQuality] artifact, which contains the demultiplexed reads from the Atacama soil microbiome tutorial.

Please select a download option that is most appropriate for your environment: 
wget \
  -O "demux.qza" \
  "https://data.qiime2.org/2024.2/tutorials/read-joining/atacama-seqs.qza"
curl -sL \
  "https://data.qiime2.org/2024.2/tutorials/read-joining/atacama-seqs.qza" > \
  "demux.qza"

Joining reads

Next, use the merge-pairs method in the q2-vsearch plugin to join the reads:

qiime vsearch merge-pairs \
  --i-demultiplexed-seqs demux.qza \
  --o-merged-sequences demux-joined.qza \
  --o-unmerged-sequences demux-unjoined.qza

Output artifacts:

Viewing a summary of joined data with read quality

You can next generate a summary of the demux-joined.qza artifact.

qiime demux summarize \
  --i-data demux-joined.qza \
  --o-visualization demux-joined.qzv

Output visualizations:

This summary is particularly useful for estimating joined read length as well as the quality scores at each sequence base position. If you hover over a specific position on the interactive quality plot you will see the table below the plot updates itself to display the parametric seven-number summary for that sequence base position. This table corresponds to what is visually represented by the box plot at that position. Between the plot and the table you can see that 10,000 out of the 40,126 sequences were used to estimate the quality scores at each position.

Hovering over positions towards the other end of the plot and examining their respective seven-number summary shows the gradual decline in quality scores that is frequently observed towards the 3’ end.

Based on the demultiplexed sequence length summary table at the bottom of this visualization, we can see that most of our sequences are at least 250 bases long. This information along with what we’ve noted about the quality scores above will help us to determine the trim length that we’ll use in the denoising step below.

Sequence quality control

Next we’ll apply quality control to our sequences using quality-filter q-score. The parameters to this method have not been extensively benchmarked on joined read data, so we recommend experimenting with different parameter settings.

qiime quality-filter q-score \
  --i-demux demux-joined.qza \
  --o-filtered-sequences demux-joined-filtered.qza \
  --o-filter-stats demux-joined-filter-stats.qza

Output artifacts:

At this stage you can choose to proceed using Deblur for additional quality control, or you can dereplicate sequences and optionally cluster them into OTUs with q2-vsearch. Deblur should give much higher quality results, so we recommend that procedure and will illustrate that approach in the next steps of this tutorial.

If you are instead interested in experimenting with an analysis workflow that is more like QIIME 1 processing (for example, to compare your Deblur or DADA2 result with a QIIME 1-like pipeline), you should next dereplicate and cluster your sequences. If you try this option, we strongly encourage you to call qiime quality-filter q-score with a higher min-quality threshold - possibly --p-min-quality 20 or --p-min-quality 30 (see Bokulich et al. 2013 for more details). You can then follow the steps in the OTU clustering tutorial. After clustering, you will likely want to filter features that are observed in only one sample using qiime feature-table filter-features --p-min-samples. In addition, removing singletons with an abundance filter is also advisable (see Bokulich et al. 2013 for more details), as well as filtering chimeric sequences.

Deblur

You’re now ready to denoise your sequences with Deblur. You should pass the sequence length value you selected from the quality score plots for --p-trim-length. This will trim all sequences to this length, and discard any sequences which are not at least this long.

Note

We use a trim length of 250 based on the quality score plots generated from the tutorial data set. Do not use 250 with your own data set, as this value will depend on your data set’s read lengths. Use the quality score plots to choose an appropriate trim length for your data.

qiime deblur denoise-16S \
  --i-demultiplexed-seqs demux-joined-filtered.qza \
  --p-trim-length 250 \
  --p-sample-stats \
  --o-representative-sequences rep-seqs.qza \
  --o-table table.qza \
  --o-stats deblur-stats.qza

Output artifacts:

View summary of Deblur feature table

You can next summarize the feature table resulting from q2-deblur. This table and the corresponding representative sequences are now ready to be analyzed with the same methods and visualizers that would be used on single-end read data.

qiime feature-table summarize \
  --i-table table.qza \
  --o-visualization table.qzv

Output visualizations:

Importing pre-joined reads

If you have joined your reads outside of QIIME 2, for example with fastq-join, this section will illustrate how to import those reads. First, download the following demultiplexed and joined read data, which has been joined on a per-sample basis with fastq-join.

Please select a download option that is most appropriate for your environment: 
wget \
  -O "fj-joined.zip" \
  "https://data.qiime2.org/2024.2/tutorials/read-joining/fj-joined.zip"
curl -sL \
  "https://data.qiime2.org/2024.2/tutorials/read-joining/fj-joined.zip" > \
  "fj-joined.zip"

Unzip this file as follows:

unzip fj-joined.zip

Import reads

Next, use qiime tools import to import these data. The format that is currently used here is SingleEndFastqManifestPhred33V2 - this will likely be updated in the future to a format that clearly describes this as joined read data, but in the meantime you should follow the formatting guidelines for the single-end “Fastq Manifest” formats.

qiime tools import \
  --input-path fj-joined/manifest \
  --output-path fj-joined-demux.qza \
  --type SampleData[JoinedSequencesWithQuality] \
  --input-format SingleEndFastqManifestPhred33V2

Output artifacts:

Viewing summary of imported data with read quality

You can generate a summary of the resulting artifact as follows:

qiime demux summarize \
  --i-data fj-joined-demux.qza \
  --o-visualization fj-joined-demux.qzv

Output visualizations:

You can now continue analyses with your joined reads as described above, e.g. quality filtering with q2-quality-filter, denoising with q2-deblur, or dereplicating and picking OTUs with q2-vsearch.

Happy QIIMEing!