Next generation sequencing

Question 1

What are the general approaches to improving the throughput/ cost ratio

Answer 1

Miniaturization, parallelization, or multiplexing

Question 2

Describe the common preparation steps of NGS

Answer 2

> The target DNA is fragmented
Common adaptors are attached to one or both ends
The results are amplified- this generates a library of short regions that cover the original sequence
the elements of the region are separated spatially (either in an array of wells or fixed to a solid medium) and sequenced in parallel (de novo sequence assembly or mapping to a reference genome.

Question 3

List the different sequencing platforms (Rapmonster’s irreplacable illuminating personality out ten/10)

Answer 3

> Roche 454 Life Sciences    2
> Ion Torrent/ Personal Genome Machine    2
> Illumina (Solexa)    2
> PacBio    3
> Oxford Nanopore    3
> The Bionano Iris system
> 10X Genomics

Question 4

Illumina (Solexa)

Answer 4

The Illumina system involves parallel simultaneous sequencing of a library of fragments, by synthesis. Successive bases are added and detected by fluorescence signals. The workflow involves:
> Generation of a library, by fragmenting the DNA sample and ligating a common adaptor to both ends. Typical fragment lengths are 300-800 bp.
> Library fragments are attached(via adaptors) to oligonucleotides on a flow-cell. A flow-cell (glass slide with lanes) can contain billions of nanowells. Each well comprises a ‘lawn’ of attached oligonucleotides that are complementary to the adaptor sequence
> bridge amplification in situ generates a monoclonal cluster of replicated fragments around each original library fragment; one cluster per nanowell
> Sequencing by synthesis: In successive cycles, the fragment clusters are exposed to a mixtures of NTPs. At each step, the polymerase adds a nucleotide. All 4 reagents contain a blocking group so that no more than 1 nucleotide is added at each step. Each of the four nucleotides has a different fluorescent tag, excited by a laser pulse. The distribution of colours, in an image of the field, identifies which base was added to each cluster. The fluorescent tags and blocking groups are removed and the cycle is repeated. The result is a kaleidoscopic movie of shifting colours, one frame per position, one colour per cluster, generating the separate sequences of library fragments.

Question 5

Explain paired-end sequencing

Answer 5

Starting from a sample of DNA, create a library of fragments and isolate those that are approximately 500bp long. Attach common adaptors to both ends of each fragment. For each fragment, sequence 50bp from each end. You now know- the nucleotide sequence of one end of the fragment, the nucleotide sequence of the other end of the fragment, and that the distance between these regions in the original sequence is about 500 bp.
You do not know the exact distance between the two reads and you do not know the sequence between the two reads.
Nevertheless, this information can help:
> if a reference sequence is available, one can detect deletions, insertions, repeats, and other structural rearrangements.
> to assemble a genome de novo, paired-end reads can bridge(link) short contigs. It is possible to ‘fill-in’ the sequence between paired-end reads, with sequences with shorter fragments that overlap the ends. Given that repetitive sequences are difficult to assemble, if one end of the fragment is part of a repeat sequence such as Long or Short Interspaced Nuclear Element (LINE or SINE), but the other is anchored in a non-repetitive sequence, you know approximately how far the repeat sequence is from the anchored one. You need additional information from the other fragment sequences to determine exactly how far apart they are.