Mouse Genome Sequencing---Sequencing strategy and the Physical Map

10/15/2002Wei Yuan

Reference:1) Gregory, et al. A physical map of the mouse genome. Nature, 2002, 418, 743-750

2) Green, ED, Strategies for the systematic sequencing of complex genomes. Nature reviews Genetics, 2001, 2, 573-583

First Part

Strategies for the systematic sequencing of complex genomes

Source: Lisa Stubbs, Lawrence Livermore National Lab

Why Mouse?

1) Human and mouse genomeshave conserved blocks ofgenetic material

Human

Mouse

http://www.informatics.jax.org

Why Mouse?

Humans and miceshare many of the

same genes

Mouse- Human Comparative Map(2 cM around Acrb gene)

Hypertension QTL in Mouse & Human (B. Paigen & G. Churchill)

Species Concordance for Susceptibility Alleles for Hypertension

Why Mouse?

Humans and micesuffer from similar

diseases

Strategies for Complex Genomes sequencing

•clone-by-clone shotgun sequencing

• whole-genome shotgun sequencing

• hybrid strategies for shotgun sequencing

Contig: overlapping series of clones or sequences reads (for a clone contig or sequencing contig, respectively) that corresponds to a contiguous segment of the source genome.

Two main shotgun-sequencing strategies

whole-genomeclone-by-clone

For clone-by-clone, a sequence-ready BAC contig map is required

Minimal Tiling Path: a minimal set of overlapping clones that together provides complete coverage across a genomic region. (The 11 clones outlined in red, which provide a minimal tiling path across the corresponding genomic region, were selected for sequencing. )

Sequence-ready BAC contig map. A collection of overlapping bacterial artificial chromosome (BAC) clones that contain human DNA was subjected to restriction enzyme digest-based fingerprint analysis. The resulting data was analysed using the program FPC, which constructed the depicted BAC contig map that spans >1 Mb.

The probability of two clones overlapping is based on the similarity of their fragments, performed by the program FPC.

FPC uses an algorithm to cluster clones into contigs based on their probability of coincidence score. For each contig, it builds a consensus band (CB) map which is similar to a restriction map; but it does not try to resolve all the errors. The CB map is used to assign coordinates to the clones based on their alignment to the map and to provide a detailed visualization of the clone overlap.

M is the number of shared bands, nL and nH are the lowest and highest number of bands in the two clones, respectively, t is

the tolerance, gellen is approximately the number of possible values, b = 2t/gellen, and p = (1 b)nH,.

two clones are considered to overlap if the following score is below a user supplied cutoff:

Shotgun-sequence assembly ---display from the program Consed

Hybrid shotgun-sequencing approach

Hybrid shotgun-sequencing approach

• take benefits of both clone-by-clone and whole-genome shotgun

• whole-genome shotgun: provides rapid insight about the sequence of the entire genome

• clone-by-clone shotgun: simplifies the process of sequence assembly to individual clone-sized genomic segments, thereby minimizing the likelihood of serious misassemblies

Used by NIH in mouse genome sequencing. (Celera is using whole genome shotgun)

Second Part

Construction of a physical map of the mouse genome

Physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA.

It provides:

• a framework for assembly of whole-genome shotgun sequence data

• a tile path of clones for generation of the reference sequence

Strategy: Using the human sequence as a framework!

Benefit:

1. Give a better level of resolution

2. Accelerate the process of constructing the mouse clone map

But why to choose human sequence?

Because they are similar in sequence organization!

• 180 conserved synteny (a region where the chromosomal location of multiple genes is conserved)

• conserved segment/linkage (a region where the order of multiple genes on a single chromosome segment is the same in both species)

Comparing Human and Mouse DNA

• Most human genes have mouse orthologs• Coding exons usually correspond 1-1• Coding sequence similarity ~ 85%

Source: Lisa Stubbs, Lawrence Livermore National Lab

Why Mouse?

1) Human and mouse genomeshave conserved blocks ofgenetic material

Human

Mouse

Let’s go back to an old slide

How to construct a physical clone map of the mouse genomeTwo Phases:

Phase I: Generation of a human-mouse homology clone map

• Compared restriction digest patterns (‘fingerprint’) of 305,716 BAC clones. Identified overlaps between clones on the basis of similarity between fingerprints and use this information to construct 7,587 contigs of overlapping clones.

----Done by the program FPC, under high strigency conditions at a probability of 1x10-16 and a match tolerance of seven

• Align the mouse BAC contigs to the human genome sequence by BES (BAC end sequences). Extend and join contigs where possible after re-examing the fingerprint data (p>1x10-12).

---- Done by BLASTN (with a blast score>700)

Phase II: Generation of a mouse clone map

Use a set of independently mapped mouse markers (available in existing genetic and radiation hybrid maps of the mouse) to position the BAC contigs in the mouse genome.

---Markers were added to the map either by electronic PCR, or by hybridization using probes

After further manual contig editing was carried out (p>1x10-10), a mouse clone map comprising 296 contigs was generated.

Construction of human–mouse homology clone map

Alignment between part of human chromosome 6 (Hsa6) and mouse chromosome 4 (Mmu4). A 1.6-Mb interval is enlarged, showing part of Hsa6q16.1 aligned to a 1.3-Mb mouse BAC contig.

11 of the 15 segments of human sequence match to 29 of the BESs within a mouse BAC contig

Summary statistics of human-mouse homology clone map

Summary statistics of mouse physical clone map by chromosome

More details about the mouse physical map

• found 51,486 homologous crosslinks btw two genomes

• Of the clones in the human genome tile path, 88% are collinear with the mouse BAC map. For individual human chromosomes, coverage by aligned mouse contigs exceeds 80% on all except chromosome 19 (61%) and the Y chromosome (0%).

• Of the total coverage of the mouse BAC map (in 211 contigs), 97% (2,658 Mb) is aligned to the human genome sequence.

• Most mouse BAC contigs contained multiple mouse markers (average 57 markers per contig).

• coverage of the mouse genome (2.8 Gb) in mapped BACs is virtually complete: 296 contigs of average size 9.3 Mb cover an estimated 2,739 Mb. (~98%)

• 275 gaps due to breaks in synteny btw the two genomes.

Now Available in Draft Form!

The Mouse Genome

http://www.ncbi.nlm.nih.govhttp://genome.ucsc.eduhttp://www.ensemble.org

• Finish sequencing by 2005

• AnalysisSequence comparisonsAnnotationGene Expression analysisGlobal genomic analysis

Future Work for Mouse Genome Sequencing