Purpose of sequence assembly
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1 Sequence Assembly
2 Purpose of sequence assembly Reconstruct long DNA/RNA sequences from short sequence reads Genome sequencing RNA sequencing for gene discovery Amplicon sequencing But not for transcript quantification Variant discovery metagenomics
3 Shear Genomic DNA
4 Sequences both ends of each fragment
5 Sequences both ends of each fragment
6 Align sequence reads to form contigs
7 Paired ends allow linking of contigs into scaffolds captured gaps scaffold In the sequence file, gaps are represented with Ns AGTCCCCTGGGAGATACGNNNNNNNNNNNNNNGATGATCAGCCGCATGAGCAG
8 Genome Assemblers
9 De Novo Genome Assembly Two major strategies: Overlap Layout Consensus Long reads > 250 bp Pairwise comparison of reads to identify overlaps Eulerian paths/de Bruin graphs Short reads 200 bp Cataloging of subsequences (k-mers) Reconstruction of paths through the k-mers
10 Overlap Layout Consensus Fragment DNA Sequence fragments Compare all sequence reads in pairwise fashion Calculate # of overlapping bases Build a matrix
11 Determine overlaps
12 Determine Layout of Overlaps Examine best overlaps: Check their layout: GCATCGTG CATCGTGA ATCGTGAT From: Computational Genome Analysis: An Introduction; Deonier et al.
13 Add new overlaps in a greedy fashion From: Computational Genome Analysis: An Introduction; Deonier et al.
14 Determine consensus sequence G From: Computational Genome Analysis: An Introduction; Deonier et al.
15 DeBruijn Graphs From Compeau et al., Nature Biotech, 2011
16 Supplementary Figures Why are de Bruijn graphs useful for genome assembly? Eulerian cycles with sequencing errors Phillip E. C. Compeau, Pavel A. Pevzner & Glenn Tesler a ATGG TGGC GGCG GCGT CGTG GTGC TGCA GCAA CAAT ATG TGG GGC GCG CGT GTG TGC GCA CAA AAT AATG b TGGA GGAG GAGT GGA GAG AGT ATGG TGGC GGCG GCGT CGTG GTGC TGCA GCAA CAAT ATG TGG GGC GCG CGT GTG TGC GCA CAA AAT AGTG c Supplementary Figure 1. De Bruijn graph from reads with sequencing errors. (a) A de Bruijn graph E on our set of reads with k = 4. Finding an Eulerian cycle is already a straightforward task, but for this value of k, it is trivial. (b) If TGGAGTG is incorrectly From Compeau et al., Nature Biotech, 2011
17 Eulerian cycle with repeated sequences CAA CA AA 13 GT AAT 14 4 CGT 8 9 AT 12 GCA CG ATG 1 3 GCG TG GTG 5 6 TGC 2 7 GC TGG 10 GG 11 GGC ATG TGC GCG CGT GTG TGC GCG CGT GTG TGG GGC GCA CAA AAT ATG Genome:ATGCGGTGCGTGGCAATG From Compeau et al., Nature Biotech, 2011 Supplementary Figure 2. De Bruijn graph of a genome with repeats. The graph E for k-mers with different multiplicities: each of the four 3-mers TGC, GCG, CGT, and GTG has multiplicity
18 Assembly reports /****************************************************************** ** ** 454 Life Sciences Corporation ** Newbler Metrics Results ** ** Date of Assembly: 2011/07/13 14:14:57 ** Project Directory: /home/ctbull2/mlf_pl3_1 ** Software Release: ( _1124) ** ******************************************************************* /* ** Input information. */ rundata file path = "/home/ctbull2/mlf_pl3_1/g5ma0n401.sff"; numberofreads = , ; numberofbases = , ; file path = "/home/ctbull2/mlf_pl3_1/g5ma0n402.sff"; numberofreads = , ; numberofbases = , ; file path = "/home/ctbull2/mlf_pl3_1/g5kxoo202.sff"; numberofreads = , ; numberofbases = , ; file path = "/home/ctbull2/mlf_pl3_1/g5sayn202.sff"; numberofreads = , ; numberofbases = , ; file path = "/home/ctbull2/mlf_pl3_1/g5sayn201.sff"; numberofreads = , ; numberofbases = , ;
19 Alignment metrics readalignmentresults file path = "/home/ctbull2/mlf_pl3_1/g5ma0n401.sff"; numalignedreads = , 99.23%; numalignedbases = , 99.56%; inferredreaderror = 0.73%, ; file path = "/home/ctbull2/mlf_pl3_1/g5ma0n402.sff"; numalignedreads = , 99.12%; numalignedbases = , 99.52%; inferredreaderror = 0.84%, ; file path = "/home/ctbull2/mlf_pl3_1/g5kxoo202.sff"; numalignedreads = , 98.90%; numalignedbases = , 99.42%; inferredreaderror = 1.01%, ; file path = "/home/ctbull2/mlf_pl3_1/g5sayn202.sff"; numalignedreads = , 98.72%; numalignedbases = , 99.26%; inferredreaderror = 1.08%, ; file path = "/home/ctbull2/mlf_pl3_1/g5sayn201.sff"; numalignedreads = , 98.87%; numalignedbases = , 99.33%; inferredreaderror = 1.05%, ;
20 Alignment metrics /* ** Consensus results. */ consensusresults readstatus numalignedreads = , 98.97%; numalignedbases = , 99.44%; inferredreaderror = 0.91%, ; numberassembled = ; numberpartial = 36389; numbersingleton = 3797; numberrepeat = 5839; numberoutlier = 5340; numbertooshort = 19103; largecontigmetrics numberofcontigs = 1457; numberofbases = ; avgcontigsize = 29101; N50ContigSize = 72969; largestcontigsize = ; Q40PlusBases = , 99.59%; Q39MinusBases = , 0.41%; allcontigmetrics numberofcontigs = 1949; numberofbases = ;
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