Shotgun DNA Sequencing: Assembly, Coverage, and Sequencing Strategies

All flashcards

What is shotgun sequencing?

A method that randomly fragments DNA, sequences the resulting pieces, and uses overlapping reads to assemble the original sequence.

What is a sequencing read?

A short sequence of bases obtained from sequencing one DNA fragment.

How are reads assembled into longer sequences?

Assembly software detects matching sections between reads, aligns those overlaps, and orders the reads to build longer composite sequences.

Why can repetitive DNA cause assembly ambiguity?

Similar or repeated sequences can make reads from different locations look alike, so software may not be able to place them uniquely.

How is average read coverage estimated?

Coverage is estimated as $C = \frac{N \times L}{G}$, where $N$ is the number of reads, $L$ is average read length, and $G$ is genome length.

Quiz questions

1. What is the typical length range of DNA strands suited to Sanger sequencing?Answer: About 100 to 1,000 base pairs.
2. What is the main purpose of hierarchical shotgun sequencing?Answer: To map large DNA fragments before sequencing smaller pieces.
3. What challenge can arise when short-read technologies produce very large numbers of reads?Answer: The large read set can make computational assembly demanding.
4. Compared with 16S rRNA amplicon sequencing, what can metagenomic shotgun sequencing help researchers do?Answer: Survey nonbacterial organisms and recover genes from mixed samples.
5. A 3,000-base genome is sequenced with 10 reads averaging 600 bases. What is the estimated coverage?Answer: $2\times$, because $\frac{10 \times 600}{3{,}000} = 2$.