# Presentation: 201915

## Whole Genome Shotgun AssemblyAlla L Lapidus, Ph.D

**    ****Solving a one-dimensional puzzle**
**    ****with millions of pieces (without the**
**             ****box)**

**(Fig:Jarrod Chapman**
**DOE_JGI)**

## Library construction

**                  ****Genome libraries **
_**Small insert library**_** **
**A small insert shotgun library consists of small DNA fragments (3 kb and 8kb) inserted into a cloning vector and maintained in a cloning host. **
**The clones should be randomly distributed and represent the whole genome. **
**During the shotgun phase of the genome project, sequences from both ends are obtained from a large number of shotgun clones. **
_**Large insert clone library (e.g. a fosmid library) **_
**The assembly of the complete genome from the individual sequences is largely facilitated when a large insert clone library (e.g. a fosmid library) is available. It helps in the gap closure phase, allows to cope with repeat sequences, and permits to validate the genome assembly. **
**End sequencing of the large insert clone library allows to map the clones onto the contigs of the genome assembly.**
**Upon analysis of mate pairs, i.e. the two ends of a single clone, bridging clones and misassembly points can be detected. **

## Genome Assembly

- contig

- 3kb

- 3kb

- 3kb

- 3kb

- 3kb

- 3kb

- 3kb

- 3kb

- 3kb

- 3kb

- ----------8kb----------

- ------------------------------------------40kb (fosmids)-----------------------------------------

**Assembly**

**Library** **Creation**

**Sequencing**

## What we get

**What we get**

## Problems: why do we have gaps

**-****a biased base content (this can result in failure to be cloned, poor stability in the chosen host-vector system, or inability of the polymerase to reliably copy the sequence). For example AT-rich DNA clones poorly in bacteria.  GC rich DNA is difficult to PCR and to sequence and often requires the use of special chemistry (sequencing kits and approaches)**
** **
**-the presence of small hairpins (inverted repeat sequences) in the DNA that re anneal ether during sequencing or electrophoresis resulting in failed sequencing reactions or unreadable electrophoresis results. (This can be aided by adding modifiers to the reaction, sequencing smaller clones and running gels at higher temperatures in the presence of stronger denaturants).**

_**What are gaps?**_
**- Genome areas not covered by random shotgun**

## Problems: why do we have gaps (cont)

**-the presence of poisonous sequence: sequence that interferes with the biology of the host organism. This could be an operator that reduced effective concentrations of an essential DNA binding regulator, or an open reading frame that expresses a toxic protein. These problems are commoner for bacterial genomes cloned in bacterial vector-host systems, but can affect eukaryotic cloning too****	****	****	****	****	****	****	****	**
**-the presence of repetitive DNA can result in deletion of sequences from the clone, particularly in bacterial systems, and thus the sequenced DNA will not correspond to the situation in the original source organism **

## Finishing/Gap Closure

**Assembly**

**Library** **Creation**

**Sequencing**

**Finishing/Gap Closure**

- contig1

- contig2

- Mis-Assembly

- Sequence gap

- Single stranded

- Low quality

- consensus

## Finishing/Gap Closure

**Assembly**

**Library** **Creation**

**Sequencing**

**Gap Closure/Finishing**

- contig1

- contig2

- Mis-Assembly

- Sequence gap

- Single stranded

- Low quality

- consensus

- -------------------------------

## Assembling repeats

**Assembling repeats**

## Finishing/Gap Closure

**Assembly**

**Library** **Creation**

**Sequencing**

**Gap Closure/Finishing**

- Low quality

- consensus

- contig1

- contig2

- Sequence gap
- (clones, PCRs)

- Single stranded

- -------------------------------

- -------------------

## Finishing/Gap Closure

**Assembly**

**Library** **Creation**

**Sequencing**

**Gap Closure/Finishing**

- Single stranded
- (clones, PCRs)

- Low quality

- consensus

- -------------------------------

- -------------------

## Finishing/Gap Closure

**Assembly**

**Library** **Creation**

**Sequencing**

**Gap Closure/Finishing**

- consensus

- -------------------------------

- -------------------

- High Quality Sequense
- < 1 error/10 000 bp

## Problems

** ****shotgun sequencing is highly automated but finishing is not: **
**most finishing decisions and manipulations**
** ****are made by people**

## A typical microbial genome sequencing project