Whole Genome Assembly
WGA
1. Screener
2. Overlapper
3. Unitigger,
4. Scaffolder,
5. Repeat Resolver.
Overlapper
...looks for end-to end overlaps of at least 40 bp with
no more than 6% differences in match.
What’s the significance?
...a one in 1017 event.
Sequencing Fidelity: 99.96%
However
...the Screener doesn’t include all of the “low
frequency” level repeats,
...so, a majority of the Overlapper outputs are bogus.
Unitigger
...differentiates between a true overlap, and an
overlap that includes more than one loci.
8X
...in a world where
real data matches
expected data, each
loci would have 8X
coverage,
...over-collapsed.
...if there were
repeats, then
contigs would be
“over-represented”,
on average 8 more
per repeat.
What Now?
... uniquely assembled contigs (unitigs) are readily
identifiable,
– all of the assembled sequences match over all of the
known sequence,
- and -
...are consistent with an 8x coverage.
Unitigs
...contig cluster is
consistent with
expected size,
...no dissimilar
sequences between
any members.
...all other contigs are sent to the Discriminator.
Discriminator
...parses the “overcollapsed” contig
by using sequence
outside of the
overlap region
Discriminator
...may yield unitigs.
Unitigger Output
...correctly assembled contigs covering 73.6%
of the genome.
Repeat Resolver
...most of the remaining gaps were due to repeats.
1. Allow “low Discriminator Value” contigs to fill gaps,
2. Find BAC sequences that unambiguously match outside the
nearest unitig,
– 1 in 107 chance of being wrong,
3. Ensure the mate end sequence of candidate BACs match.
If that Doesn’t Work
...find a mate-pair that spans the gap, and sequence
it,
...make sequencing
primer from BES...
Chromosome Walking
Scaffolder
...contigs the contigs,
– uses mate-pair information.
WGA Result
...91% sequence, 9% gaps,
Compartmentalized Shotgun Assembly
Mapping
Scaffolds
Sequence Tagged Sites
STS
...PCR primers are designed for unique regions of
the genome or chromosome,
...the chromosome is cut ,
...assay two PCR products, frequency of coamplification indicates .
Sequence Tagged Sites
STS
Compartmentalized Shotgun Assembly
...ideally 24,
...really 3845.
CSA
92.2 % Sequence
7.8 % Gaps
WPA
91 % Sequence
9 % Gaps
Chromosome 21
Blue: Gaps
Violations:
Red : misoriented
Yellow: distance
PFP
CSA
Green: Same Order,
Orientation
Yellow: Same
Orientation
Red: Out of Order,
Orientation
Chromosome 8
PFP
CSA
PFP
CSA
Major Public Sequence Databases
• 281 Curated Data Bases,
• “... facilitating Biological Discovery”.
What Do We Know?
(based on functional group analysis)
Science 291 (5507), 1304-1351
Functional Groups
1st GenBank NR protein database was partitioned into clusters using BLASTP,
Describing Aligned Sequences
2
nd
Statistical descriptions of the cluster are developed and tested,
• Hidden-Markov Markers: statistical descriptions of aligned sequences.
Functional Group Annotation
3
rd
Categorization was done by manual review of the family and
subfamily names,
...by examining SwissProt and GenBank records,
...and by review of the literature as well as resources on the World
Wide Web.
http://www.expasy.ch/cgi-bin/niceprot.pl?P29965
Outcomes?
• A relatively small number of structural and functional
domains are used in a large number of different proteins,
• Pfam: 527 families,
• average length is 275 residues,
• 456 had “annotated functions”.
Nucleic Acids Research 26, 320-322
New Genes
4
th
Newly sequenced genes are virtually translated, and the
predicted proteins are assayed against raw and HMM databases,
...significance cut-off levels are determined for each functional group
family.