Hi everyone. I have paired end sequencing data and need to produce a metadata tsv for my QIIME2 scripts. Does anyone have an example of one for paired end sequencing data?
I have forward and reverse barcodes so do i need to combine these for my barcode column?
Best wishes
Hi @Mantella86,
Do you need to import the samples in QIIME2? If so you probably need a manifest file, which
links sample names and fastq file paths. All the process is described at:
https://docs.qiime2.org/2021.4/tutorials/importing/#sequence-data-with-sequence-quality-information-i-e-fastq
If you scroll down at the ' “Fastq manifest” formats', the first example is what you need
Once you created your manifest file, the command to use will be something like:
qiime tools import
--type 'SampleData[SequencesWithQuality]'
--input-path your-manifest.tab
--output-path paired-end-demux.qza
--input-format PairedEndFastqManifestPhred33V2
That is if you have 2 fastq files for each sample, if you have only 2 fastq files including all samples, we need to know more information on the library type. For some, it may be possible to import as above and demultiplex within QIIME2, for other you will need to demultiplex outside QIIME2 and import them as above after this step.
If you already imported your sample in QIIME2 and you need metadata file for the analysis, you can avoid to list the barcodes at this point, and there will be no difference between single or paired end data, and example is:
https://data.qiime2.org/2021.4/tutorials/moving-pictures/sample_metadata.tsv
Cheers
Luca
Hi Luca, thank you for your reply. I have imported my barcodes at the start in the form off PairedEndFastqManifestPhred33V2 so later on when i import the metadata file are you saying i can leave out the barcodes?
There are 2 fastq files for each sample as it is forward and reverse.
Kind regards
Hi @Mantella86,
In the metadata there is one row for each sample, if it is derived from PE or not does not matter.
In most of the analysis you are interested in group comparisons, so you may leave out the barcodes which would act as unique identifier for the sample. Some visualiser it may even complain saying that there are only group composed by 1 sample only.
That said, sometimes you may want to look at per sample alpha-diversity. For that, a unique identifier such as the barcode is exactly what you need to have in the metadata file fed to the alpha-diversity visualiser.
Hope it helps
Luca
Thank you Luca. To make it one row per sample then i will need to combine my forward and reverse barcodes into one, is that correct or just leave them separate?
Hi @Mantella86,
I usually like to keep them separate by '-', as:
ATCACGAC-TGAACCTT
(Just an example, these barcodes my not be used at all in your case!)
Cheers
Luca
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