Sequence an-alysis Work flows

. Sequence analysis with Stata. The Stata Journal, 6(4), 435-460. -PDF- 生命历程调查 (Life History Analysis) 最近几年在社会学中越来越火热。

Sequence analysis Work flows

• experiment design
  • RNA-seq
    • Known gene / transcript diffential expression
    • Novel transcript discovery
    • Single- versus paired-end
      • Most analysis now paired-end
      • Reads within exons
      • A single end spanning exons: ‘junction reads’
      • Reads spanning exons
  • ChIP-seq
  • Variants
    • Germline vs. somatic
    • Exome vs. whole genome
    • SNP vs. indel vs. structural
  • Copy number
    • Low vs. high coverage
• MeRIP sequencing
  • General processes
    • 1. m6A specific immunoprecipitation
      • 1. fragmentation of mRNA
      • 2. apply antibodies against m6A
    • 2. next generation sequencing
  • m6A methylation
    • mRNA N6-methyladenosine (m6A) methylation
    • on mRNA, A, and the 6 position (nitrogen)
  • input control sequences
    • IP
      • immunoprecepeted small mRNA segments
    • Input
      • The original mRNAs
• Sequencing
  • a picture
  • 100’s of millions of reads
  • 30-150 nucleotides
  • Single and paired-end
  • Bar codes, lanes & flow cells
• Analysis
  • DNA
  • RNA
  • epigenetics
  • integrative
  • microbiome
• Quality assessment
  • Assessment, eg. fastqc
    • Read length, duplication
    • Nucleotide use: per cycle, GC content, N content, . . .
    • Quality: per cycle, per read
    • Consistency across samples, without obvious treatment-specific associations
  • Remediation, e.g., trimmomatic
    • Crop e.g., leading / tailing artifacts of sequencing protocol
    • Trim based on quality
• Fastq
  • Anntoation
    • machine, flow cell, tile, coordinates, end
    • @ERR127302.1709 HWI-EAS350\_0441:1:1:1461:7837\#0/1
  • DNA sequence
    • usually A, C, T, G, N (uncalled)
    • CAGCCACAGAACCACGGCACGGAAGACATGAGGCAGCATGCTCACGAGA...
  • Quality (approximately, − log10(p))
    • encoded as ASCII
      • Different encodings
      • characters: 40 is approximately p = 0.0001, 30 is p = 0.001,
      • Higher in the alphabet is better
    • HHGHHHH>DH:@.7@49;88G8>G>DDG@D>D@G@GE>@DDBDDG<A82...