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I am looking at the videos at a DIY Transcriptomics course and the speaker mentions that to run Kallisto for read alignment with paired end sequencing, one would enter the following:

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where the sample files at the end refer to the two different end reads (forward and reverse read files). However, browsing the NCBI for paired end data, I noticed that only 1 Fastq file is provided per replicate for a paired end procedure (here's an example). So I'm a bit confused about this.

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Building off of the answer by @An Ignorant Wanderer:

Essentially there are indeed the paired end data files available, but it takes a little bit of extra navigation to reach it.

Using the specific example you provided of BioProject PRJNA574273: First select a specific sequencing "Run," aka the sequencing of a specific sample / experimental condition. For example, click on the link to run: SRR10189234

This brings you to the default tab: "Metadata." As @An Ignorant Wanderer pointed out, then you can navigate to the "Data Access" tab.

Then look under the "Original format" subheading. Here you'll find links to two files, corresponding to the two orientations of the paired end sequence files.

Edit: Also please see the answer by @burger, the website linked in that answer is much more user friendly in my opinion.

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The problem is that the SRA website can be a little confusing. View the same project on ENA where the organization is more human-readable: https://www.ebi.ac.uk/ena/browser/view/PRJNA574273

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Actually after clicking on one of the "Runs" and going to "data access" I noticed two files are provided per run

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Paired ends is a configuration in sequencing platforms. Illumina is the most popular at the moment and most single-cell sequencing is done using paired-end Illumina sequencing. It varies between single-cell technologies but most 3’ scRNA-Seq protocols use paired ends.

Kallisto is a tool for handling raw data. Many databases supported processed data in BAM format or UMI/read counts. You should not need to run kallisto unless you are involved in handling raw data from sequencing performed by your laboratory. Some databases such as SRA and EGA support raw data in FASTQ format, others do not for ethical reasons or the data is too large.

I recommend downloading from SRA via their command line interface (this is documented on their website). It is possible to extract paired ends from an aligned BAM file using bedtools and samtools. I’ve done this with the Macosko et al (Cell 2015) DropSeq data for example.

Note that in some cases, the barcode is stored in databases as the headers of the FASTQ, BAM, BED, or CTSS file rather than a separate read. Single cell transcriptomics is an emerging field so there isn’t a standard way to do this, it varies between labs and data centres. For single-cell specifically, some databases are being set up, for example: https://data.humancellatlas.org/

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