How to import one fastq file （all sample data were demultiplexed and merged into one fastq file）

[yuhe (yuhexiang)]

I plan to analyze Illumina Paired-end Sequencing data by using qiime2. However, these datas were sequenced three years ago, and forward and reverse reads have been merged, and barcodes has been removed, even demultiplex samples have been merged into a fastq file, and I wonder how to imports this fastq file by using qiime2. This question has puzzled me for a long time, and I really hope it can be solved. Thank you!

[thermokarst]

@yuhe

demultiplex samples have been merged into a fastq file,

Can you correct me if I am wrong - it sounds like you are demultiplexing and the multiplexing the same data. I am not sure what that would accomplish for you, but for most things in QIIME 2 you will need to have demultiplexed data. If your reads are already joined (forward and reverse combined) and demultiplexed, you can use the fastq-manifest format to import SampleData[JoinedSequencesWithQuality]:

qiime tools import \
 --type 'SampleData[JoinedSequencesWithQuality]' \
 --input-path se-33-manifest.csv \
 --output-path single-end-demux.qza \
 --source-format SingleEndFastqManifestPhred33

Then, you can use q2-deblur or q2-vsearch to process these data (you won't be able to use q2-dada2, because it operates on unjoined paired-end reads). Let us know if you have any questions!

[yuhe (yuhexiang)]

thermokarst，
Thank you very much for your reply. All reads were already joined (forward and reverse combined), demultiplexed and merged into one file and its format is as follows：

but when executing this command“qiime tools import --type 'SampleData[JoinedSequencesWithQuality]' --input-path QY1-48.new.fastq --output-path single-end-demux.qza --source-format SingleEndFastqManifestPhred33 ”, The following mistake is shown :“There was a problem importing QY1-48.new.fastq:

QY1-48.new.fastq is not a(n) SingleEndFastqManifestPhred33 file
”

[thermokarst]

Hi @yuhe, I am still a bit confused about this part of your description:

@yuhe

demultiplexed and merged into one file

What does that mean? Demultiplexing means to separate one file into per-sample files, so it seems confusing that you would multiplex these data after demultiplexing, but maybe I am misunderstanding.

@yuhe

QY1-48.new.fastq is not a(n) SingleEndFastqManifestPhred33 file

Please double-check the link I provided above, the tutorial provides a complete example of how to create a fastq-manifest file, and then import it (hint --- your --input-path parameter should point to your fastq-manifest file, not a fastq file).

Let us know how it goes!