<p>I received the following message from the author of another Drop-seq paper:</p>

<blockquote>
  <p>After much conversation with the folks at the SRA, it appears the SRA
  compression algorithm automatically deletes certain subsets of bam
  tags and does so in cryptic way (in the sense of not alerting the
  uploader). Obviously this is going to be an issue for many single cell
  datasets encoded this way, and I hope the SRA will eventually change
  their system to accommodate.</p>
</blockquote>

<p>If you look at the "Download" tab of the <a href="https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR6261587" rel="nofollow noreferrer">same page</a>, there now exists a link to download the originally uploaded BAM file.</p>

<p>Alternately, you can use the <a href="https://www.ncbi.nlm.nih.gov/sra/docs/sra-aws-download/" rel="nofollow noreferrer">SRA AWS tutorial</a> to download the files on that platform. </p>

CC BY-SA 4.0
2019-07-29T23:19:55.283Z
2019-07-29T23:19:55.283Z
Jim Notwell
<p>In case of Drop-seq, we have paired end data.</p>

<p>Read 1: Cell code + UMI (unique molecule identifier)</p>

<p>Read 2: The transcript information</p>

<p>But I have a problem/doubt with the sample I am working on.</p>

<p>The sample I am using is the <a href="https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR6261587" rel="nofollow noreferrer">following</a> (Check the "Reads" tab).</p>

<p>As you know the Drop-seq is "paired-end", we are expected to see two reads for a spot. Although this sample say paired-end, it has only one read per spot.</p>

<p>For example I can share a link of a different scRNA-seq data where you can properly see two reads for a spot</p>

<p><a href="https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR8086553" rel="nofollow noreferrer">Example sample</a>, you need to check the "Reads" tab.</p>

<p>Where I am going wrong?</p>

<p>I asked one of the main authors of the paper. The following is the reply I got :</p>

<blockquote>
  <p>I recommend that you download the aligned BAM files that are hosted in
  the same GEO record. Read 1 is already processed into the cell and UMI
  barcodes and held as custom tags (XC and XM) in the BAM files. The
  cells are already barcode-corrected, so if you use those files, your
  cell barcodes will line up with mine; if you start from FASTQs, they
  will not. For most aligners, you can just use the BAM file as input to
  realign. (It has all reads, even those that did not align.)</p>
</blockquote>

<p>But I could not find any "XM" or "XC" keywords in the bam file :(</p>

<p>To understand his reply you have to be familiar the <a href="https://github.com/broadinstitute/Drop-seq/files/2425535/Drop-seqAlignmentCookbookv1.2Jan2016.pdf" rel="nofollow noreferrer">processing steps of the Drop-seq</a>.</p>

<p>Looks like they have submitted some kind of processed data. I could not figure out how much the data is processed. I am trying to use the data starting at different processing steps but I could not figure out how much the data is processed.</p>

CC BY-SA 4.0
2018-11-13T09:43:47.187Z
Where did you even find their BAM files in GEO? I only see the output from CellRanger (UMI counts and such).
Devon Ryan
CC BY-SA 4.0
2018-11-13T10:28:23.440Z
My guess is the authors uploaded BAM files to GEO which have been converted back to fastq in SRA, losing all the extra annotation.
Ian Sudbery
<scrnaseq><read-mapping>
Problem: "Pair-end" reads scRNA seq data (Drop-seq)
# Problem: "Pair-end" reads scRNA seq data (Drop-seq)

(Asked by: username_0 on 2018-11-13)

In case of Drop-seq, we have paired end data.

Read 1: Cell code + UMI (unique molecule identifier)

Read 2: The transcript information

But I have a problem/doubt with the sample I am working on.

The sample I am using is the [following](https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR6261587) (Check the "Reads" tab).

As you know the Drop-seq is "paired-end", we are expected to see two reads for a spot. Although this sample say paired-end, it has only one read per spot.

For example I can share a link of a different scRNA-seq data where you can properly see two reads for a spot

[Example sample](https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR8086553), you need to check the "Reads" tab.

Where I am going wrong?

I asked one of the main authors of the paper. The following is the reply I got :

> I recommend that you download the aligned BAM files that are hosted in
> the same GEO record. Read 1 is already processed into the cell and UMI
> barcodes and held as custom tags (XC and XM) in the BAM files. The
> cells are already barcode-corrected, so if you use those files, your
> cell barcodes will line up with mine; if you start from FASTQs, they
> will not. For most aligners, you can just use the BAM file as input to
> realign. (It has all reads, even those that did not align.)

But I could not find any "XM" or "XC" keywords in the bam file :(

To understand his reply you have to be familiar the [processing steps of the Drop-seq](https://github.com/broadinstitute/Drop-seq/files/2425535/Drop-seqAlignmentCookbookv1.2Jan2016.pdf).

Looks like they have submitted some kind of processed data. I could not figure out how much the data is processed. I am trying to use the data starting at different processing steps but I could not figure out how much the data is processed.

**Tags:** scrnaseq, read-mapping
> **Comments:**
> **username_1**: Where did you even find their BAM files in GEO? I only see the output from CellRanger (UMI counts and such).  
> **username_2**: My guess is the authors uploaded BAM files to GEO which have been converted back to fastq in SRA, losing all the extra annotation.  

---

## 1 answers

### Answer by username_3 on 2019-07-29. Score: 3

I received the following message from the author of another Drop-seq paper:

> After much conversation with the folks at the SRA, it appears the SRA
> compression algorithm automatically deletes certain subsets of bam
> tags and does so in cryptic way (in the sense of not alerting the
> uploader). Obviously this is going to be an issue for many single cell
> datasets encoded this way, and I hope the SRA will eventually change
> their system to accommodate.

If you look at the "Download" tab of the [same page](https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR6261587), there now exists a link to download the originally uploaded BAM file.

Alternately, you can use the [SRA AWS tutorial](https://www.ncbi.nlm.nih.gov/sra/docs/sra-aws-download/) to download the files on that platform.


---

