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lecture_notes:04-08-2015

De novo assembly II

Guest lecturer: Stefan Prost, stefan.prost@berkley.edu

Illumina paired-end sequencing libraries

  • MiSeq has 300 bp reads
  • Paired ends read from both directions, so you get one read for each end (may or may not overlap depending on molecule and read size)
	=====> end_1
	________________________
	           end_2 <=====
  • Problem: not really sufficient for repetitive regions
    • ends can't be very far apart because Illumina can't handle big molecules
    • not enough info for scaffolding

Illumina mate-pair sequencing libraries

  • Idea: get paired reads that are much farther away (for more scaffolding info)
  • Basically, the same idea as paired-ends, except the middle section is missing and the ends are oriented the opposite way.
		<===== end_1
	________________________
	           end_2 =====>
  • Genomic DNA > Fragment (2-5 kb) > biotinylate ends > Circularize > Fragment (400-600 bp) > Enrich biotinylated fragments > Ligate adaptors > …
    • Cut DNA, attach a biotin tag to both ends of the target molecule
    • Circularize target molecule
      • This step is hard
    • Circularized molecule is fragmented, fragment with the biotin tag (a.k.a the ends of the original target stuck to each other backwards, with the tags in between) is enriched
    • Then end repair, A-tailing, adapters added, amplification, sequencing
  • Dependent on inferring insert size (can be tricky)
  • Most companies can get you 8 kb inserts, with skill you can get up to 20 kb
  • Complicated process, weird stuff can happen in between
  • Important difference between paired ends and mate-pairs: ends are oriented the opposite way.

BAC (Bacterial Artificial Chromosome) and fosmid libraries

Read quality assessment

  • Base quality: Phred scores reported by sequencer.
  • Fastq files: fasta files, plus encoded phred scores
    • Need to know if your file has phred33 or phred64 encoding
  • Quality for each individual base is not the whole story, the context matters to the signal processing too
  • Reads decrease in quality further down the read
    • Depending on the assembler, you might need to trim lower quality reads off the end. Others want untrimmed data
  • Pacific Bio doesn’t have GC|AT bias

Tools

  • FastQC (Most popular tool to tell you about the read library)
    • FastQC reported an issue with our data with kmer count (related to adapter content)
      • This needs to be checked out and diagnosed!
  • Preqc
    • Estimates how difficult the assembly will be

Estimating genome size from read data

G = (pn(1-k+1))/(λ_k)
G = Genome size
pn = proportion of correct reads
k = kmer length
λ_k= mode of the k-kmer count histogram
Simpson 2013, arXiv
  • To estimate genome size need to know:i) total number of reads; ii) length of reads; and iii) kmer distribution

Error correction

  • High amount of small kmers are usually errors

Simulated contig length in the k-de Brujin graph can estimate the best kmer to use for assembly. Based on contig length N50

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lecture_notes/04-08-2015.txt · Last modified: 2015/04/17 15:34 by sihussai