Answers.Body: <p>I recommend that you look at the most recent version of the protocol, which includes example adapter and barcode files:</p>
  
  <p><a href="https://www.protocols.io/view/stranded-mapping-from-long-reads-syheft6?step=2" rel="nofollow noreferrer">https://www.protocols.io/view/stranded-mapping-from-long-reads-syheft6?step=2</a></p>
  
  <p>[check in the protocol to see if there's a more recent version]</p>
  
  <p>These files contains the public sequences that ONT have released for their sequencing kits (as briefly mentioned in the "Before Starting" section of the protocol). The sequences are available on the ONT community, but I have converted them into FASTA files for use in my protocol.</p>
  
  <p>The adapter and barcode files were combined into a single FASTA file in the previous version of my protocol. For the PCR cDNA barcoding, it looked something like this:</p>
  
  <pre><code>&gt;ONT_VNP
  ACTTGCCTGTCGCTCTATCTTCTTTTT
  &gt;ONT_SSP
  TTTCTGTTGGTGCTGATATTGCTGCCATTACGGCCGGG
  &gt;BC01
  AAGAAAGTTGTCGGTGTCTTTGTG
  &gt;BC02
  TCGATTCCGTTTGTAGTCGTCTGT
  &gt;BC03
  GAGTCTTGTGTCCCAGTTACCAGG
  &gt;BC04
  TTCGGATTCTATCGTGTTTCCCTA
  &gt;BC05
  CTTGTCCAGGGTTTGTGTAACCTT
  &gt;BC06
  TTCTCGCAAAGGCAGAAAGTAGTC
  &gt;BC07
  GTGTTACCGTGGGAATGAATCCTT
  &gt;BC08
  TTCAGGGAACAAACCAAGTTACGT
  &gt;BC09
  AACTAGGCACAGCGAGTCTTGGTT
  &gt;BC10
  AAGCGTTGAAACCTTTGTCCTCTC
  &gt;BC11
  GTTTCATCTATCGGAGGGAATGGA
  &gt;BC12
  CAGGTAGAAAGAAGCAGAATCGGA
  &gt;RB12A
  GTTGAGTTACAAAGCACCGATCAG
  </code></pre>
  
  <p>I separate out adapters and barcodes in the newer versions of the protocol because I was getting too many incorrect hits.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-12-27T09:09:56.780Z
Answers.LastActivityDate: 2018-12-27T09:09:56.780Z
Answers.UserDisplayName: gringer
Body: <p>I recently started doing the analysis on nanopore sequencing data. As I was searching for some help on pre-processing of the data, I found your nice setup pipeline created here: <a href="https://www.protocols.io/view/stranded-mapping-from-long-reads-n8cdhsw" rel="nofollow noreferrer">https://www.protocols.io/view/stranded-mapping-from-long-reads-n8cdhsw</a>.</p>
  
  <p>As I was trying to follow the instruction, I ran into a question on part where it uses lastal to divide the input file as strand specific output.</p>
  
  <p>Part that uses the following script below:</p>
  
  <pre><code>lastal -P 10 ONT_barcodes_adapters.fa &lt;(zcat 4T1_BC06.correctedReads.uniqueOnly.fasta.gz) | \  ~/scripts/maf_bcsplit.pl | grep -e 'ONT_SSP,+' -e 'ONT_VNP,-' | \   awk -F',' '{print <span class="math-container">$1}' | sort | uniq &gt; fwdQry_seqs_BC06.txt lastal -P 10 barcodes_primerSeqs.fa &lt;(zcat 4T1_BC06.correctedReads.uniqueOnly.fasta.gz) | \   ~/scripts/maf_bcsplit.pl | grep -e 'ONT_SSP,-' -e 'ONT_VNP,+' | \   awk -F',' '{print $</span>1}' | sort | uniq &gt; revQry_seqs_BC06.txt 
  </code></pre>
  
  <p>In here, I was wondering what the ONT_barcodes_adapters.fa file actually looks like. I wanted to find the information in other places and could not find it elsewhere.</p>
  
  <p>If you could help me with this, I would really appreciate it!</p>
  
Tags: <nanopore>
Title: Question on nanopore sequencing data process pipeline (cDNA-PCR)
ThreadText: # Question on nanopore sequencing data process pipeline (cDNA-PCR)
  
  (Asked by: username_0 on 2018-12-26)
  
  I recently started doing the analysis on nanopore sequencing data. As I was searching for some help on pre-processing of the data, I found your nice setup pipeline created here: <https://www.protocols.io/view/stranded-mapping-from-long-reads-n8cdhsw>.
  
  As I was trying to follow the instruction, I ran into a question on part where it uses lastal to divide the input file as strand specific output.
  
  Part that uses the following script below:
  
  ```
  lastal -P 10 ONT_barcodes_adapters.fa <(zcat 4T1_BC06.correctedReads.uniqueOnly.fasta.gz) | \  ~/scripts/maf_bcsplit.pl | grep -e 'ONT_SSP,+' -e 'ONT_VNP,-' | \   awk -F',' '{print $1}' | sort | uniq > fwdQry_seqs_BC06.txt lastal -P 10 barcodes_primerSeqs.fa <(zcat 4T1_BC06.correctedReads.uniqueOnly.fasta.gz) | \   ~/scripts/maf_bcsplit.pl | grep -e 'ONT_SSP,-' -e 'ONT_VNP,+' | \   awk -F',' '{print $1}' | sort | uniq > revQry_seqs_BC06.txt 
  
  ```
  
  In here, I was wondering what the ONT\_barcodes\_adapters.fa file actually looks like. I wanted to find the information in other places and could not find it elsewhere.
  
  If you could help me with this, I would really appreciate it!
  
  **Tags:** nanopore
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2018-12-27. Score: 1 | Accepted answer
  
  I recommend that you look at the most recent version of the protocol, which includes example adapter and barcode files:
  
  <https://www.protocols.io/view/stranded-mapping-from-long-reads-syheft6?step=2>
  
  [check in the protocol to see if there's a more recent version]
  
  These files contains the public sequences that ONT have released for their sequencing kits (as briefly mentioned in the "Before Starting" section of the protocol). The sequences are available on the ONT community, but I have converted them into FASTA files for use in my protocol.
  
  The adapter and barcode files were combined into a single FASTA file in the previous version of my protocol. For the PCR cDNA barcoding, it looked something like this:
  
  ```
  >ONT_VNP
  ACTTGCCTGTCGCTCTATCTTCTTTTT
  >ONT_SSP
  TTTCTGTTGGTGCTGATATTGCTGCCATTACGGCCGGG
  >BC01
  AAGAAAGTTGTCGGTGTCTTTGTG
  >BC02
  TCGATTCCGTTTGTAGTCGTCTGT
  >BC03
  GAGTCTTGTGTCCCAGTTACCAGG
  >BC04
  TTCGGATTCTATCGTGTTTCCCTA
  >BC05
  CTTGTCCAGGGTTTGTGTAACCTT
  >BC06
  TTCTCGCAAAGGCAGAAAGTAGTC
  >BC07
  GTGTTACCGTGGGAATGAATCCTT
  >BC08
  TTCAGGGAACAAACCAAGTTACGT
  >BC09
  AACTAGGCACAGCGAGTCTTGGTT
  >BC10
  AAGCGTTGAAACCTTTGTCCTCTC
  >BC11
  GTTTCATCTATCGGAGGGAATGGA
  >BC12
  CAGGTAGAAAGAAGCAGAATCGGA
  >RB12A
  GTTGAGTTACAAAGCACCGATCAG
  
  ```
  
  I separate out adapters and barcodes in the newer versions of the protocol because I was getting too many incorrect hits.
  
  
  ---
  
  