1. Virginia Commonwealth University
Genome Research
Ping Xu
For BBSI
The Philips Institute
Virginia Commonwealth University
Richmond, Virginia

2. Microbial genome projects in Virginia Commonwealth University
1. Cryptosporidium hominis
2. Streptococcus sanguis
3. Trypanosoma cruzi
4. Human BAC or cosmid clones
5. Bacterial phages

5. General Procedures in Sequencing
Subclone
(genomic shotgun library)
Production sequencing
Template isolation
Sequencing reactions
Fragment separation
Data acquisition
Base calling
Finishing
Assembly
Gap filling
Conflict resolution
Verification
Analysis
Gene predictions
Homology searches
Annotation

6. PE/ABI 3700 Capillary Sequencer
- automated
- faster runs (8 per day)
- capillary (easy to use)
- 96 capillaries per run
- $300,000 per machine
Truly automated High Throughput sequencing

7. Raw data from ABI 3700 Prism Sequencer
2/9/2018
Raw data from ABI 3700 Prism Sequencer

8. Both
strand
Gap
Single
clone
Gap
Single
strand

10. Strategies to sequence a genome
Whole genome shotgun sequence
Whole genome mapping based
Hybrid above two strategies

11. From Rob Martienssen
Cold Spring Harbor Laboratory

12. Hybrid strategy Assemble shotgun sequences Alignment with BAC clones
Overlay with other genomes
Blast search

13. Filtering
Way to remove a large percentage of the repetitive regions of the genome under whole genome sequencing.
Methyl filtration is one approach
Physical methods may also be useful (hybridization methods)

14. Skimming 1. Carry out 1-3 fold coverage of the region
2. Can be whole genome or clone based
4. Covers ~66 – 97% of the target sequence
5. 99% or grater accuracy on average
3. Clone based can therefore be targeted
1. Carry out 1-3 fold coverage of the region

15. Rough draft 1. Typically 5X coverage 2. Can be thought of as:
High coverage skimming
Low coverage complete sequencing
3. Advantages and disadvantages are intermediate between skimming and complete sequencing
4. Some are proposing 10X rough draft as “finished”

16. Complete Sequence 1. More than 10X coverage All base accurate
2. Finishing
Assembly
Gap filling
Conflict resolution
Verification
3. Analysis
Gene predictions
Homology searches
Annotation

17. Goals of Complete Genome project
1. Complete filling gaps
2. Complete finishing
3. The base accuracy: 1 error/10 K
4. Complete annotation

18. Locating and filling the gaps
1. Find the shotgun or BAC sequence pairs to bridge the gaps
2. Comparison with other closely related finished genomes
3. Blast search to find the hits over two contigs
4. Re-sequence shotgun clones with short sequences to extent the contigs
5. Design primers for genome walking
6. Multiple PCR to orient contigs for no-hit contigs and PCR amplification to bridge the gaps.

19. Finishing
Finishing is the process of assembling and refining raw sequence data into a highly accurate final genomic sequence
1. Automated sequence editing
2. Manual, interactive sequence inspection
3. Directed sequencing
4. Assembly verification
5. Remove contaminated sequences

20. High Accuracy of Sequence
1. Diagnostics, forensics, etc.
2. Protein coding predictions
3. Repeat sequence, polymorphisms, SNPs, etc.
4. Evolution analysis and phylogenics

21. How do we analyze sequence once obtained for gene functions?
Computational analysis
Database searches (DNA or protein)
Compositional and domain analysis
Comparative genomics
etc.
“Wet lab”
Individual gene analysis
Chip analysis
Knock out

22. Function Conservation
Homology searches
Biochemical function
The function almost always conserved between homologous
Physiology function
The phenotypes related to conserved orthologous proteins
3. The biochemical function can be reliable inferred from genetic homology. The physiology function cannot.

23. 2/9/2018

24. 2/9/2018

25. Sequence processing Basecalling + Quality trimming with Phred
2/9/2018
Sequence processing
Basecalling + Quality trimming with Phred
Phred quality score:
10 means 1 error in 10
20 means 1 error in 100
30 means 1 error in 1000
40 means 1 error in 10000

26. A common program for sequence assembling and finishing
Phred/Phrap/Consed
A common program for sequence assembling and finishing

27. Phred
Phred is a base-calling program for DNA sequence traces. The program was developed by Drs. Phil Green and Brent Ewing. It is widely used by the largest academic and commercial sequencing laboratories.

28. Phrap
Phrap is a leading program for DNA sequence assembly. Phrap is routinely used in some of the largest sequencing projects in the Human Genome Sequencing Project and in the biotech industry.

29. Consed
Consed is a graphical tool for sequence finishing. It is a program for editing sequence assemblies created with Phrap assembly program. In addition to a full set of standard features (view traces, edit reads by inserting a base, deleting a base, substituting a base, etc.), it supports an efficient editing procedure designed for use by Phrap in subsequent reassemblies of the same data set.

30. 2/9/2018

31. 2/9/2018

32. 2/9/2018