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application_of_pacbio_long_reads_sequencing_technology [2018/11/29 12:41] – [phh5tools] hyjeongapplication_of_pacbio_long_reads_sequencing_technology [2021/03/17 13:09] (current) – external edit 127.0.0.1
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   * [[https://github.com/PacificBiosciences/Bioinformatics-Training/wiki|PacBio bioinformatics training wiki]]   * [[https://github.com/PacificBiosciences/Bioinformatics-Training/wiki|PacBio bioinformatics training wiki]]
  
-===== phh5tools =====+===== pbh5tools =====
 A Swiss-army knife for interrogating PacBio HDF5 files (cmp.h5, bas.h5) A Swiss-army knife for interrogating PacBio HDF5 files (cmp.h5, bas.h5)
- 
   * https://github.com/PacificBiosciences/pbh5tools   * https://github.com/PacificBiosciences/pbh5tools
   * https://github.com/PacificBiosciences/pbh5tools/blob/master/doc/index.rst (사용법)   * https://github.com/PacificBiosciences/pbh5tools/blob/master/doc/index.rst (사용법)
  
---readType은 ccs, subreads, unrolled. ccs는 bas.h5 파일 내부에 ccs read가 있는 경우에 뽑아낸다. unrolled는 어떤 것인지 잘 모르겠다.+  * **bash5tools.py** can extract read sequences and quality values for both Raw and circular consensus sequencing (CCS) readtypes and use create fastq and fasta files. 
 +  * **cmph5tools.py**는 PacBio Alignment File Format(cmp.h5, [[https://pacbiofileformats.readthedocs.io/en/3.0/legacy/CmpH5Spec.html|링크]]) 파일을 다루는 도구라는데 나는 아질 쓸 일이 없다. 
 + 
 +--readType은 ccs, subreads, unrolled. ccs는 bas.h5 파일 내부에 ccs read가 있는 경우에 뽑아낸다. unrolled는 어떤 것인지 잘 모르겠다. 이것이 바로 raw read 그대로를 의미하는 것일까?
  
   $ bash5tools.py input.bas.h5 --outFilePrefix myreads --outType fasta --readType subreads --minReadScore 0.75   $ bash5tools.py input.bas.h5 --outFilePrefix myreads --outType fasta --readType subreads --minReadScore 0.75
  
 +Analysis Results 서브디렉토리에 있는 p0.[1-3].subreads.fast{a|q} 파일의 수치와 bash5tools.py를 이용해서 bas.h5 파일로부터 추출한 read의 수치를 비교해 보았다. 2번 항목부터 bash5tools.py를 이용한 것이다.
 +
 +  - subreads.fastq 전체: 1339659706 bp / 124615 seqs; 10750.4 average length
 +  - (--readType unrolled): 1395878244 bp / 101548 seqs; 13746.0 average length
 +  - (--readType subreads): 1394529365 bp / 131741 seqs; 10585.4 average length
 +  - (--readType subreads --minReadScore 0.7): 1347690522 bp / 126271 seqs; 10673.0 average length
 +  - (--readType subreads **--minReadScore 0.75**): 1339662407 bp / 124756 seqs; 10738.3 average length
 +  - (--readType subreads --minReadScore 0.8): 1287559618 bp / 118740 seqs; 10843.5 average length
 +  - (--readType subreads --minReadScore 0.85): 1038658565 bp / 92346 seqs; 11247.5 average length
 +  - (--readType subreads --minReadScore 0.9): 654807 bp / 121 seqs; 5411.6 average length
 +  - (--readType subreads --minReadScore 0.95): no sequence extracted!
 +
 +--minReadScore가 0.9에 근접하면서 결과물의 분량이 현저히 떨어진다. --minReadScore 0.75로 하는 것이 Analysis Results 서브디렉토리에 있는 subreads file의 분량과 거의 흡사하다.
 ===== SMRT analysis ===== ===== SMRT analysis =====
   * SMRT analysis system requirements [[http://www.pacb.com/wp-content/uploads/2015/09/SMRT-Analysis-System-Requirements.pdf|PDF 문서]]   * SMRT analysis system requirements [[http://www.pacb.com/wp-content/uploads/2015/09/SMRT-Analysis-System-Requirements.pdf|PDF 문서]]
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