<p>Building off of the answer by @An Ignorant Wanderer:</p>

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

<p>Using the specific example you provided of BioProject <a href="https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA574273&amp;o=acc_s%3Aa" rel="nofollow noreferrer">PRJNA574273</a>:
First select a specific sequencing "Run," aka the sequencing of a specific sample / experimental condition. For example, click on the link to run: <a href="https://trace.ncbi.nlm.nih.gov/Traces/sra?run=SRR10189234" rel="nofollow noreferrer">SRR10189234</a> </p>

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

<p>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.</p>

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

CC BY-SA 4.0
2020-06-09T17:18:58.560Z
2020-06-12T13:11:03.340Z
2020-06-12T13:11:03.340Z
Dan
<p>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: <a href="https://www.ebi.ac.uk/ena/browser/view/PRJNA574273" rel="nofollow noreferrer">https://www.ebi.ac.uk/ena/browser/view/PRJNA574273</a></p>

CC BY-SA 4.0
2020-06-09T18:48:12.743Z
2020-06-09T18:48:12.743Z
burger
<p>Actually after clicking on one of the "Runs" and going to "data access" I noticed two files are provided per run </p>

CC BY-SA 4.0
2020-06-09T15:03:29.427Z
2020-06-09T15:03:29.427Z
An Ignorant Wanderer
<p>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.</p>

<p>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.</p>

<p>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. </p>

<p>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: <a href="https://data.humancellatlas.org/" rel="nofollow noreferrer">https://data.humancellatlas.org/</a></p>

CC BY-SA 4.0
2020-06-13T06:50:59.357Z
2020-06-13T06:56:56.723Z
2020-06-13T06:56:56.723Z
Tom Kelly ケリー・トム
<p>I am looking at the videos at a <a href="http://diytranscriptomics.com/" rel="nofollow noreferrer">DIY Transcriptomics course</a> and the speaker mentions that to run Kallisto for read alignment with paired end sequencing, one would enter the following:</p>

<p><a href="https://i.sstatic.net/3w2hf.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/3w2hf.png" alt="enter image description here"></a></p>

<p>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 (<a href="https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA574273&amp;o=acc_s%3Aa" rel="nofollow noreferrer">here</a>'s an example). So I'm a bit confused about this. </p>

<rna-seq><kallisto>
Files for paired end RNA sequencing
# Files for paired end RNA sequencing

(Asked by: username_0 on 2020-06-09)

I am looking at the videos at a [DIY Transcriptomics course](http://diytranscriptomics.com/) and the speaker mentions that to run Kallisto for read alignment with paired end sequencing, one would enter the following:

[![enter image description here](https://i.sstatic.net/3w2hf.png)](https://i.sstatic.net/3w2hf.png)

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](https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA574273&o=acc_s%3Aa)'s an example). So I'm a bit confused about this.

**Tags:** rna-seq, kallisto

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## 4 answers

### Answer by username_1 on 2020-06-09. Score: 0 | Accepted answer

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](https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA574273&o=acc_s%3Aa):
First select a specific sequencing "Run," aka the sequencing of a specific sample / experimental condition. For example, click on the link to run: [SRR10189234](https://trace.ncbi.nlm.nih.gov/Traces/sra?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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### Answer by username_2 on 2020-06-09. Score: 1

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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### Answer by username_0 on 2020-06-09. Score: 0

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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### Answer by username_3 on 2020-06-13. Score: 0

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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