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qiime tools import \

--type ‘SampleData[PairedEndSequencesWithQuality]’ \

--input-format PairedEndFastqManifestPhred33 \

--input-path data \

--output-path artifacts/xxxx.qza

The artifacts created by the above commands are demultiplexed and they are ready for

processing by QIIME2.

7.3.1.2  Metadata

In the above, we discussed importing the raw sequence data into QIIME2 artifacts. But we

have also mentioned that a sample metadata describing the study is required for the analy-

sis. A metadata file can also be created by the user and will be used later in the analysis to

show the sample grouping. A metadata file is a tab-separated value (TSV) file containing

sample and study design information in columns and the first column must be the sample

identifiers for the IDs of the sample in the study. The top line is for the column names. The

ID column name must be one of those: id, sampleid, sample id, sample-id, featured, fea-

ture id, or feature-id. The IDs listed in the metadata files should be unique and not empty.

The ID column is not optional and the metadata file must contain at least one ID. The ID

column can be followed by additional columns defining metadata associated with each

sample or feature such as groups, treatment, factor, and barcode sequence. The column

name should not be one of the reserved words and the type of the data contained in the

metadata file is either categorical or numeric. The sample metadata is created by the user

manually. However, if the data is downloaded from the NCBI SRA database, the metadata

can also be downloaded and modified to meet our goal. This will be discussed below in the

worked example.

7.3.2  Demultiplexing

Above, we discussed the EMP and non-EMP-multiplexed FASTQ files and how they can

be imported into QIIME2 artifacts. Since the reads are multiplexed, demultiplexing must

be carried out before reads processing and analysis. QIIME2 uses “demux” and “cutadapt”

plugins for demultiplexing EMP-multiplexed reads and non-EMP-multiplexed reads,

respectively.

FIGURE 7.2  Examples of manifest files for single-end and paired-end FASTQ files.