weixin_39744230
weixin_39744230
2020-12-09 14:25

None barcode

Hi,

I have a very puzzling demultiplexing problem which I have no idea why it occurs.

I have a couple of nanopore reads with bc1, bc2, bc3 but when I run the entire set, all go to the "None" barcode section. So I went down and downsampled the reads:


head -n 1300 nanpore_run2.fastq > test.fastq

Now when I run it, it will detect the following barcodes and demultiplex/trim based on those:


singularity exec ~/.singularity/porechop.img porechop -i test.fastq -v 1 -t 16 --barcode_threshold 50 -b mux

Trimming adapters from read ends
     SQK-NSK007_Y_Top: AATGTACTTCGTTCAGTTACGTATTGCT
  SQK-NSK007_Y_Bottom: GCAATACGTAACTGAACGAAGT
     1D2_part_2_start: CTTCGTTCAGTTACGTATTGCTGGCGTCTGCTT
       1D2_part_2_end: CACCCAAGCAGACGCCAGCAATACGTAACT
             BC01_rev: CACAAAGACACCGACAACTTTCTT
                 BC01: AAGAAAGTTGTCGGTGTCTTTGTG
             BC02_rev: ACAGACGACTACAAACGGAATCGA
                 BC02: TCGATTCCGTTTGTAGTCGTCTGT
             BC03_rev: CCTGGTAACTGGGACACAAGACTC
                 BC03: GAGTCTTGTGTCCCAGTTACCAGG
                 BC01: AAGAAAGTTGTCGGTGTCTTTGTG
             BC01_rev: CACAAAGACACCGACAACTTTCTT
                 BC02: TCGATTCCGTTTGTAGTCGTCTGT
             BC02_rev: ACAGACGACTACAAACGGAATCGA
                 BC03: GAGTCTTGTGTCCCAGTTACCAGG
             BC03_rev: CCTGGTAACTGGGACACAAGACTC
           NB01_start: AATGTACTTCGTTCAGTTACGTATTGCTAAGGTTAACACAAAGACACCGACAACTTTCTTCAGCACCT
             NB01_end: AGGTGCTGAAGAAAGTTGTCGGTGTCTTTGTGTTAACCTTAGCAATACGTAACTGAACGAAGT
           NB02_start: AATGTACTTCGTTCAGTTACGTATTGCTAAGGTTAAACAGACGACTACAAACGGAATCGACAGCACCT
             NB02_end: AGGTGCTGTCGATTCCGTTTGTAGTCGTCTGTTTAACCTTAGCAATACGTAACTGAACGAAGT
           NB03_start: AATGTACTTCGTTCAGTTACGTATTGCTAAGGTTAACCTGGTAACTGGGACACAAGACTCCAGCACCT
             NB03_end: AGGTGCTGGAGTCTTGTGTCCCAGTTACCAGGTTAACCTTAGCAATACGTAACTGAACGAAGT

325 / 325 (100.0%)

299 / 325 reads had adapters trimmed from their start (13,934 bp removed)
205 / 325 reads had adapters trimmed from their end (5,183 bp removed)


Discarding reads containing middle adapters
325 / 325 (100.0%)

47 / 325 reads were discarded based on middle adapters


Saving trimmed reads to barcode-specific files

  Barcode  Reads  Bases   File
  BC01        41  15,980  mux/BC01.fastq
  BC02        78  34,759  mux/BC02.fastq
  BC03        47  20,117  mux/BC03.fastq
  none       112  50,420  mux/none.fastq

Not a great yield, but still stuff is happening. Now I add 25 reads more - it detects the same set of barcodes but all of a sudden, all are labelled as "None".


head -n 1400 tcseq_nanpore_run2.fastq > test.fastq
singularity exec ~/.singularity/porechop.img porechop -i test.fastq -v 1 -t 16 --barcode_threshold 50 -b mux

Trimming adapters from read ends
     SQK-NSK007_Y_Top: AATGTACTTCGTTCAGTTACGTATTGCT
  SQK-NSK007_Y_Bottom: GCAATACGTAACTGAACGAAGT
     1D2_part_2_start: CTTCGTTCAGTTACGTATTGCTGGCGTCTGCTT
       1D2_part_2_end: CACCCAAGCAGACGCCAGCAATACGTAACT
             BC01_rev: CACAAAGACACCGACAACTTTCTT
                 BC01: AAGAAAGTTGTCGGTGTCTTTGTG
             BC02_rev: ACAGACGACTACAAACGGAATCGA
                 BC02: TCGATTCCGTTTGTAGTCGTCTGT
             BC03_rev: CCTGGTAACTGGGACACAAGACTC
                 BC03: GAGTCTTGTGTCCCAGTTACCAGG
                 BC01: AAGAAAGTTGTCGGTGTCTTTGTG
             BC01_rev: CACAAAGACACCGACAACTTTCTT
                 BC02: TCGATTCCGTTTGTAGTCGTCTGT
             BC02_rev: ACAGACGACTACAAACGGAATCGA
                 BC03: GAGTCTTGTGTCCCAGTTACCAGG
             BC03_rev: CCTGGTAACTGGGACACAAGACTC
           NB01_start: AATGTACTTCGTTCAGTTACGTATTGCTAAGGTTAACACAAAGACACCGACAACTTTCTTCAGCACCT
             NB01_end: AGGTGCTGAAGAAAGTTGTCGGTGTCTTTGTGTTAACCTTAGCAATACGTAACTGAACGAAGT
           NB02_start: AATGTACTTCGTTCAGTTACGTATTGCTAAGGTTAAACAGACGACTACAAACGGAATCGACAGCACCT
             NB02_end: AGGTGCTGTCGATTCCGTTTGTAGTCGTCTGTTTAACCTTAGCAATACGTAACTGAACGAAGT
           NB03_start: AATGTACTTCGTTCAGTTACGTATTGCTAAGGTTAACCTGGTAACTGGGACACAAGACTCCAGCACCT
             NB03_end: AGGTGCTGGAGTCTTGTGTCCCAGTTACCAGGTTAACCTTAGCAATACGTAACTGAACGAAGT

350 / 350 (100.0%)

324 / 350 reads had adapters trimmed from their start (14,999 bp removed)
220 / 350 reads had adapters trimmed from their end (5,627 bp removed)


Discarding reads containing middle adapters
350 / 350 (100.0%)

58 / 350 reads were discarded based on middle adapters


Saving trimmed reads to barcode-specific files

  Barcode  Reads  Bases    File
  none       292  127,150  mux/none.fastq

How is that possible?

该提问来源于开源项目:rrwick/Porechop

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