Next Generation Sequencing (+ Fasta & Fastq Computer Lab Session)

First Generation Sequencing = [blank_start]Sanger Sequencing[blank_end] Second Generation Sequencing = [blank_start]Next Generation Sequencing[blank_end] Third Generation Sequencing = [blank_start]Single Molecule Sequencing[blank_end]

The complete set of genes or genetic material present in a cell or organism is known as the [blank_start]genome[blank_end]

Plasmids are taken into account when considering the genome of a certain bacteria.

Sanger Sequencing - Step 1: Denature double stranded DNA into single strands using [blank_start]heat[blank_end]

Sanger Sequencing - Step 2: Single strand of DNA is then amplified via PCR

Sanger Sequencing - Step 3: A DNA [blank_start]primer[blank_end] is then attached to each of the single strands of DNA

Sanger Sequencing - Step 4: The DNA with bound primers are then added equally to [blank_start]four[blank_end] separate solutions before three types of [blank_start]deoxynucleotides[blank_end] and one type of [blank_start]dideoxynucleotide[blank_end] is added.

DNA polymerase is also added to such solutions thus allowing both deoxynucleotides and dideoxynucleotides to bind in a [blank_start]5' --> 3'[blank_end] manner

The dideoxynucleotides in each of the four solutions (i.e. one of A, C, T or G) selectively bind to the single stranded DNA within the solutions. The binding of a dideoxynucleotide stops the further binding of complementary deoxynucleotides further down the chain due to its lack of [blank_start]an -OH[blank_end] group at its [blank_start]3'[blank_end] end.

The lack of the hydroxyl group at the 3' end of the dideoxynucleotide means that no [blank_start]phosphodiester[blank_end] bond can be formed thus causing the DNA polymerase to cease extension of the complementary DNA strand from the template strand.

Sanger Sequencing - Step 5: The [blank_start]complementary[blank_end] strands of DNA are then denatured from their [blank_start]template[blank_end] strands and the latter are washed away.

Sanger Sequencing - Step 6: The DNA samples are then separated based on size via gel [blank_start]electrophoresis[blank_end] in order to determine the order of base pairs in the original sequence.

Sanger sequencing has a poor quality in the first 15-40 bases due to poor [blank_start]primer[blank_end] binding.

Sanger sequencing quality increases as the sequence goes on, with the highest quality found at the end of the sequence.

Sanger Sequencing can only be used for short DNA strands of 100 to 1000 base pairs. Thus a method whereby longer DNA strands are broken up into shorter ones is typically used, in which the DNA is broken up randomly into numerous small segments which are sequenced using Sanger sequencing to obtain a series of 'reads', which are then recombined by computer programs based on overlapping ends of reads. This method of breaking up DNA for Sanger Sequencing is known as [blank_start]shotgun[blank_end] sequencing

[blank_start]Next Generation Sequencing[blank_end] is mainly preferred to [blank_start]Sanger Sequencing[blank_end] as it can sequence many reactions in parallel where the former can only sequence one at a time, meaning the overall process is far faster for larger DNA strands.

The type of Next Generation Sequencing most commonly used (aka the one we should learn most about) is called:

Next Generation Sequencing - Step 1: The first step is Tagmentation, whereby the long strand of DNA is ligated by enzymes called [blank_start]Transposases[blank_end] into shorter segments ready for amplification.

Next Generation Sequencing - Step 2: Step 2 involves reduced cycle amplification, whereby primer binding sequences, indices and terminal sequences are added. [blank_start]Primer binding sequences[blank_end] allow the binding of the primer to the DNA strand so sequencing can occur. [blank_start]Terminal Sequences[blank_end] allow the binding of the DNA strand to the flow cell for sequencing, as they bind to complementary oligonucleotides (known as oligos) on the flow cell which thus holds them in place. [blank_start]Indices[blank_end] are the sequences used to identify samples. They are normally 6 bases long and allow each sample to be uniquely identified.

Added during reduced cycle amplification, the indices added to the strand allow for [blank_start]96[blank_end] different samples to be run together.

Next Generation Sequencing - Step 3: The DNA strands must now be amplified by a process called [blank_start]bridge[blank_end] amplification. This involves DNA polymerases first creating a complementary strand, before the original is then washed away. Following this, the reverse strand (complementary strand) which has been left bends over and attaches to the complementary [blank_start]oligo[blank_end]. DNA polymerases then create a strand identical to the original, before both are linearised for sequencing.

At the end of clonal amplification, all of the [blank_start]reverse[blank_end] strands are washed off leaving only the [blank_start]forward[blank_end] strands for sequencing.

Next Generation Sequencing - Step 4: Following clonal amplification, [blank_start]primers[blank_end] are added to the forward DNA strands which allows DNA polymerase to begin to add fluorescently tagged nucleotides to the strand. Each time one of the tagged nucleotides binds to the strand, a specific wavelength of light is given off which is then read and processed by a computer program. The details further to this are in too much depth.

NGS sequencing results tend to decrease in quality towards the end of the sequence

In Next Generation Sequencing, due to the sequencing by synthesis process if the blocker of a fluorescently tagged nucleotide is not removed following its detection, it causes a problem in the next cycle meaning that that one strand is one base behind in terms of sequencing. Throughout the sequencing process, these add up leading to a progressively decreasing quality of results as the sequencing approaches its end point. This process is typically known as [blank_start]Phasing[blank_end]

An example of third generation sequencing is minION sequencing. How does this work?

The following questions are based on the fasta/fastq tutorial which was given in the computer suite following the sequencing lecture. Click 'True'.

[blank_start]Fastq[blank_end] files start with an @ sign [blank_start]Fasta[blank_end] files start with a > sign

Fast[blank_start]q[blank_end] files have four lines, whereby the bottom line assesses [blank_start]quality[blank_end]

Poor quality of read due to phasing usually occurs at the [blank_start]3'[blank_end] end of a sample

A fragment of a genome sequence which is derived by assembling short sections of sequenced DNA into larger constructs is called a [blank_start]Contig[blank_end]

Building a Contig from smaller sequenced segments is based on identifying overlap between the sequence reads of such smaller segments.

Contigs joined together is called a [blank_start]scaffold[blank_end]

The [blank_start]N50[blank_end] statistic defines the contig length at which, when added to all larger contigs, give a total which exceeds 50% of the total assembly length. (see description for example)

The [blank_start]NG50[blank_end] statistic defines the contig length at which, when added to all larger contigs, give a total which exceeds 50% of the total estimated/reference genome

Difference between N50 and NG50: The [blank_start]N50[blank_end] statistic defines the contig length at which, when added to all larger contigs, give a total which exceeds 50% of the total assembly length. The [blank_start]NG50[blank_end] statistic defines the contig length at which, when added to all larger contigs, give a total which exceeds 50% of the total estimated genome