text: user10782: I am trying to understand how data that was uploaded to SRA (https://www.ncbi.nlm.nih.gov/sra?LinkName=biosample_sra&from_uid=4510743) can be analyzed with the assumption that the FASTQ file should contain replicate information (3 replicates within the sample). I downloaded the file and ran it with Nextflow, and it was mapped to the human genome and determined to contain unstranded reads of 50bp, both directions.
  Which steps can I take to infer if the FASTQ file indeed is not demultiplexed?
  Many thanks,
  Geo
  The header of the FASTQ looks like this:
  @SRR3467208.1 1/1
  NTCTCTATGTCCACTCTGGAGCCTTTAAGTGCCACTTGAGGGCCTTTAAC
  +
  #4:D;DDEHHFHHIJJIJJGHGHJJI@HFIIJJHJJIG?9CFHIIIHHHG
  @SRR3467208.2 2/1
  NGTGCTGTGTGTGCATGTGTGTGCGTGTGTGTGCTGTGCGTTTGTGTGTG
  +
  #1:B:BDDFFFFFFFBGFHIFIIFGFFGFGFGIIIIIIEFGGIIFFIFII
  @SRR3467208.3 3/1
  NGTCACTTGGCATCTGGCCATCGGGCTGGATGCCGTGTTCCAGGCAGTAG
  +
  #1:A=DDDB<<CFAFGI@FGG9A@68CEG;DB?<DGGGE49?BBF2B=FF
  @SRR3467208.4 4/1
  NTCCGGGGCTCCAGCAACCAGAAGAGGGAAAAGCTGTCTTCGGTGATGTC
  +
  
  
  user2108: I would suggest looking at sample metadata:
  
  Library:
  
  
  Name: TruSeq RNA
  
  
  Instrument: Illumina HiSeq 2000
  
  
  Strategy: RNA-Seq
  
  
  Source: TRANSCRIPTOMIC
  
  
  Selection: PolyA
  
  
  Layout: PAIRED
  
  You can see that it's paired. You can look at the spot descriptor at the page you link to see more information about the design.
  There is no multiplexing information available there. I would instead look at the overall project and its metadata to find the different replicates. It looks like there are 2 samples x 3 replicates each, deposited independently. So it is likely to already be demultiplexed, and you can just download them individually.
  "Unstranded" isn't really a meaningful term for this, if I understand correctly. Forward and reverse read pairs don't correspond to forward and reverse on the chromosome, but to the way that sequencing-by-synthesis happens on the machine. So the mapping orientation is not really relevant.
  I infer that you are working from a single FASTQ file. If you are working from a single FASTQ file for paired-end out of SRA, then the FASTQ file is not yet resolved into F+R reads. I would suggest rerunning the FASTQ conversion with --split-3 or similar option.
  
original_text: 10782: <p>I am trying to understand how data that was uploaded to SRA (<a href="https://www.ncbi.nlm.nih.gov/sra?LinkName=biosample_sra&amp;from_uid=4510743" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/sra?LinkName=biosample_sra&amp;from_uid=4510743</a>) can be analyzed with the assumption that the FASTQ file should contain replicate information (3 replicates within the sample). I downloaded the file and ran it with Nextflow, and it was mapped to the human genome and determined to contain unstranded reads of 50bp, both directions.</p>
  <p>Which steps can I take to infer if the FASTQ file indeed is not demultiplexed?</p>
  <p>Many thanks,
  Geo</p>
  <p>The header of the FASTQ looks like this:</p>
  <pre><code>@SRR3467208.1 1/1
  NTCTCTATGTCCACTCTGGAGCCTTTAAGTGCCACTTGAGGGCCTTTAAC
  +
  #4:D;DDEHHFHHIJJIJJGHGHJJI@HFIIJJHJJIG?9CFHIIIHHHG
  @SRR3467208.2 2/1
  NGTGCTGTGTGTGCATGTGTGTGCGTGTGTGTGCTGTGCGTTTGTGTGTG
  +
  #1:B:BDDFFFFFFFBGFHIFIIFGFFGFGFGIIIIIIEFGGIIFFIFII
  @SRR3467208.3 3/1
  NGTCACTTGGCATCTGGCCATCGGGCTGGATGCCGTGTTCCAGGCAGTAG
  +
  #1:A=DDDB&lt;&lt;CFAFGI@FGG9A@68CEG;DB?&lt;DGGGE49?BBF2B=FF
  @SRR3467208.4 4/1
  NTCCGGGGCTCCAGCAACCAGAAGAGGGAAAAGCTGTCTTCGGTGATGTC
  +
  </code></pre>
   2108: <p>I would suggest looking at sample metadata:</p>
  <blockquote>
  <p>Library:</p>
  </blockquote>
  <blockquote>
  <p>Name: TruSeq RNA</p>
  </blockquote>
  <blockquote>
  <p>Instrument: Illumina HiSeq 2000</p>
  </blockquote>
  <blockquote>
  <p>Strategy: RNA-Seq</p>
  </blockquote>
  <blockquote>
  <p>Source: TRANSCRIPTOMIC</p>
  </blockquote>
  <blockquote>
  <p>Selection: PolyA</p>
  </blockquote>
  <blockquote>
  <p>Layout: PAIRED</p>
  </blockquote>
  <p>You can see that it's paired. You can look at the spot descriptor at the page you link to see more information about the design.</p>
  <p>There is no multiplexing information available there. I would instead look at the <a href="https://www.ncbi.nlm.nih.gov/sra?term=SRP074191" rel="nofollow noreferrer">overall project and its metadata</a> to find the different replicates. It looks like there are 2 samples x 3 replicates each, deposited independently. So it is likely to already be demultiplexed, and you can just download them individually.</p>
  <p>&quot;Unstranded&quot; isn't really a meaningful term for this, if I understand correctly. Forward and reverse read pairs don't correspond to forward and reverse on the chromosome, but to the way that sequencing-by-synthesis happens on the machine. So the mapping orientation is not really relevant.</p>
  <p>I infer that you are working from a single FASTQ file. If you are working from a single FASTQ file for paired-end out of SRA, then the FASTQ file is not yet resolved into F+R reads. I would suggest rerunning the FASTQ conversion with <code>--split-3</code> or similar option.</p>
  