index

Videos are available in the very last section "Auxiliaries".

Subjects to be discussed;

1) When using short-read with high numbers of reads; when using longer-reads with lower numbers?

2) what is Paired-end sequencing ?

3) NGS sequencing of very short RNAs (e.g. micro-RNAs, tRNAs etc.)

4) "exome" sequencing and "Targeted" sequencing

5) How does Pacific Bio long-run sequencing work ?

6) How does Nanopore sequencing work ?

7) other (your proposals)

Next-generation sequencing (NGS) is a massively parallel sequencing technology that has revolutionized the biological sciences. With its ultra-high throughput, scalability, and speed, NGS enables researchers to perform a wide variety of applications and study biological systems at a level never before possible.  The most important innovations of NGS regard the (1) lack of DNA/RNA fragment cloning, (2) the use of micro- or nano-reactors immobilized on distinct solid supports, enabling a very high level of parallelization of in situ sequencing and (3) the absence of electrophoretic separation of fragments because once nucleotides are incorporated in the sequencing reaction they are simultaneously identified.

To be analyzed the DNA is fragmented with chemical or enzymatic methods in defined fragments obtaining library of fragments that are subjected to covalent link with adaptators and subsequently used for the clonal amplification and sequencing.

The length of the reads can be different depending on the NGS technology exploited: Illumina’ reads length is around 100/150, for Abi SOLiD is 85, for 454 is around 700, exc. Short reads are mainly used of the re-sequencing when  mapping against a reference genome; whereas longer reads are used for de novo sequencing, starting for primary data lacking a genome of reference.

The paired-end sequencing allows users to sequence both ends of a fragment: after sequencing using a primer for a specific strand of it, the following step uses the opposite primer to sequence the anti-strand. It generates high-quality, alignable sequence data.  Moreover, it facilitates detection of genomic rearrangements (as insertions, deletions and inversions) and repetitive sequence elements, as well as gene fusions and novel transcripts. Paired-end DNA sequencing reads provide superior alignment across DNA regions containing repetitive sequences, and produce longer contigs for de novo sequencing by filling gaps in the consensus sequence.

The contigs can in turn be ordered to form scaffold on the basis of information of connectivity provided by the pairs of sequences that derive from the ends of the same clone (paired-end). In fact, if the two portions of a paired-end sequence map on two different contigs it is possible that those contigs are contiguous in the genome.