Question

1. In a nutshell, what are the major steps performed during next generation sequencing of DNA? How do these steps differ in the Illumina platform as compared to the Roche 454 platform?

2. In a nutshell, describe the major steps performed during DNA sequencing by the Pacific Biosciences, as well as the Oxford Nanopore Sequencing Technologies.

3. In a nutshell, describe the major advantages and disadvantages of each DNA sequencing platform.

Answer 1

1)

Diagramatic reperesentation of the preparation of library as well as process of sequencing of the most frequently used next generation sequencing methods.

Summary of the Roche 454 sequencing platform. DNA breaks into smaller fragments, where adapters are ligated, fragments are linked to beads, combined into an emulsion, emulsion PCR amplification, placed in picotitre plate's well, then estimate the sequences by pyrosequenicng.

2)

We can detect DNA methylation in unaltered DNA by using Pacific Biosciences SMRT technology and Oxford Nanopore third generation sequencing.

3)

Next Generation Platform	Main advantages	Main disadvantages
Roche 454	Generate maximum read length	Expensive, high cost
Illumina	Suitable for whole genome sequencing, low cost, high throughput.	Lagging strand dephasing leads to the poor quality of read at the end.
Pacific Biosciences	Real time sequencing through single molecule approach generates maximum number of reads, low run time,	High cost, high error rate, low total number of reads
Oxford Nanopore	Low cost, long reads, minion is a usb device	Unknown error rates
Gilbert and sanger method of traditional sequencing	In Gilbert method, use of radioactive labels In Sanger method, fluorescent dye used for labelling.	Slow, high cost per run.