Question

Could you please provide me the answer for following multiple choice question- subject- Genes, Genomics and Human Health( Genetics)

Question-2

Question 2.1

Whole genome DNA sequencing can be applied to genome-wide association studies to

		sequence the genomes of as many as 100,000 individuals
		search for rare Mendelian coding mutations
		search for very rare variants on haplotypes identified in genome-wide genotyping studies
		identify family pedigrees in large populations

Question 2.2

Next Generation sequencing aims to sequence the genome to a coverage of

		5-10X
		10-20X
		a minimum of 30X
		a minimum of 30-50X

Question 2.3

Hybridization of the DNA primer to a single-stranded DNA template allows

		binding of RNA polymerase and extension of the template strand from the free 3'-OH group
		transcription of RNA
		binding of DNA polymerase and extension of the template strand from the free 3'-OH group
		binding of DNA polymerase and extension of the template strand from the free 5'-OH group

Question 2.4

• The second step in a Next Generation DNA sequencing reaction is

		hybridization of DNA fragments via ligated adapters to the flow cell
		bridge amplification of hybridized genomic DNA to generate clusters of cloned DNA
		fragmentation of the genomic DNA and ligation of adapters
		cycle sequencing of all amplified genomic DNA clusters

Question 2.5

Next generation DNA sequencing is generally NOT used for

		parrallel sequencing of millions of short DNA fragments across the genome
		targeted sequencing of a specific gene region or locus
		sequencing of all exons in the human genome
		RNA sequencing

Question 2.6

Sanger DNA sequencing is is used for

		parrallel sequencing of millions of short DNA fragments across the genome
		targeted sequencing of a specific gene region or locus
		sequencing of all exons in the human genome
		RNA sequencing

Question 2.7

The human reference genome is

		different for each modern human population
		contains all rare deleterious mutations
		a composite genome derived from several anonymous donors
		a composite genome derived from several anonymous African donors

Question 2.8

The third step in a Next Generation DNA sequencing reaction is

		hybridization of DNA fragments via ligated adapters to the flow cell
		bridge amplification of hybridized genomic DNA to generate clusters of cloned DNA
		fragmentation of the genomic DNA and ligation of adapters
		cycle sequencing of all amplified genomic DNA clusters

Question 2.9

Coverage is

		a measure of the success of the hybridization of the genomic DNA to the flow cell
		the average number of reads representing a given nucleotide in the reconstructed sequence
		the average gene copy number in a genome
		the proportion of genes of the genome that are transcribed in a cell

Question 2.10

Whole exome sequencing can sequence more than 95% of all exons in the human genome with a coverage of

		30-50X
		100-200X
		200-500X
		500-1000X

Question 2.11

GRHL2 is a transcription factor that when mutated causes Dyskeratosis congenita. The mutations in the gene are found in the domain that

		catalyses the hydrolysis of GTP to GDP
		binds to DNA
		remodels chromatin
		acetylates histone 3, lysing residue 27

Question 2.22

The fourth step in a Next Generation DNA sequencing reaction is

		hybridization of DNA fragments via ligated adapters to the flow cell
		bridge amplification of hybridized genomic DNA to generate clusters of cloned DNA
		fragmentation of the genomic DNA and ligation of adapters
		cycle sequencing of all amplified genomic DNA clusters

Whole exome sequencing identifies exons by

		sequencing mature mRNA
		using oligonucleotides to specifically remove introns
		using oligonuceotides that specically remove non-coding DNA
		using oligonucleotides that specifically capture and recover exon-containing genomic DNA

Answer 1

2.1 Whole genome DNA sequencing(WGS) can be applied to genome-wide association studies to search for very rare variants on haplotypes identified in genome-wide genotyping studies.

In genetics, a genome-wide association study (GWA study, or GWAS), also known as whole genome association study (WGA study, or WGAS), is an observational study of a genome-wide set of genetic variants in different individuals to see if any variant is associated with a trait.

A GWAS approach can identify many known variants and replicable novel associations using whole genome sequence (WGS). For example, many scientific research results provide a clear proof of concept on the use of GWAS within HIV and other viruses whole genome sequence data.

2.2  Next Generation sequencing aims to sequence the genome to a coverage of 10× to 20×

For detecting human genome mutations, SNPs, and rearrangements,publications often recommend from 10× to 20× depth of coverage, depending on the application and statistical model.

Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies including:

• Illumina (Solexa) sequencing
• Roche 454 sequencing
• Ion torrent: Proton / PGM sequencing
• SOLiD sequencing

These recent technologies allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such have revolutionised the study of genomics and molecular biology.

x coverage (or -fold covergae is used to describe the sequencing depth. For example, if your genome has a size of 10 Mbp and you have 100 Mbp of sequencin data that is assembled to said 10 Mbp genome, you have 10x coverage.

2.3 Hybridization of the DNA primer to a single-stranded DNA template allows binding of DNA polymerase and extension of the template strand from the free 3'-OH group

A primer is a short single strand of RNA or DNA (generally about 18-22 bases) that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3′-end of the primer, and copies the opposite strand.

The DNA replication fork. RNA primer labeled at top

2.4 The second step in a Next Generation DNA sequencing reaction is bridge amplification of hybridized genomic DNA to generate clusters of cloned DNA

Next generation methods of DNA sequencing have three general steps:

• Library preparation: libraries are created using random fragmentation of DNA, followed by ligation with custom linkers
• Amplification: the library is amplified using clonal amplification methods and PCR
• Sequencing: DNA is sequenced using one of several different approaches

Amplification

Library amplification is required so that the received signal from the sequencer is strong enough to be detected accurately. With enzymatic amplification, phenomena such as 'biasing' and 'duplication' can occur leading to preferential amplification of certain library fragments. Instead, there are several types of amplification process which use PCR to create large numbers of DNA clusters.

Emulsion PCR

Emulsion oil, beads, PCR mix and the library DNA are mixed to form an emulsion which leads to the formation of micro wells (Figure 1).

Figure 1 | Emulsion PCR

In order for the sequencing process to be successful, each micro well should contain one bead with one strand of DNA (approximately 15% of micro wells are of this composition). The PCR then denatures the library fragment leading two separate strands, one of which (the reverse strand) anneals to the bead. The annealed DNA is amplified by polymerase starting from the bead towards the primer site. The original reverse strand then denatures and is released from the bead only to re-anneal to the bead to give two separate strands. These are both amplified to give two DNA strands attached to the bead. The process is then repeated over 30-60 cycles leading to clusters of DNA. This technique has been criticised for its time consuming nature, since it requires many steps (forming and breaking the emulsion, PCR amplification, enrichment etc) despite its extensive use in many of the NGS platforms. It is also relatively inefficient since only around two thirds of the emulsion micro reactors will actually contain one bead. Therefore an extra step is required to separate empty systems leading to more potential inaccuracies.

Bridge PCR

The surface of the flow cell is densely coated with primers that are complementary to the primers attached to the DNA library fragments (Figure 2). The DNA is then attached to the surface of the cell at random where it is exposed to reagents for polymerase based extension. On addition of nucleotides and enzymes, the free ends of the single strands of DNA attach themselves to the surface of the cell via complementary primers, creating bridged structures. Enzymes then interact with the bridges to make them double stranded, so that when the denaturation occurs, two single stranded DNA fragments are attached to the surface in close proximity. Repetition of this process leads to clonal clusters of localised identical strands. In order to optimise cluster density, concentrations of reagents must be monitored very closely to avoid overcrowding.

Figure 2 | Bridging PCR