Genomics Flashcards

Question

What is a contig ?

Answer 1

DNA sequence built up from a number of smaller overlapping sequences during a sequencing project.

Answer 2

is a technique used to link together a non-contiguous series of genomic sequences into a scaffold, consisting of sequences separated by gaps of known length.

Answer 3

the number of reads that include a given nucleotide in the reconstructed sequence

Answer 4

each sequenced piece of a genome

Answer 5

Hierarchal shotgun sequencing | Whole genome shotgun sequencing

Answer 6

``` DNA extraction Library construction Random sequencing phase Gap closure phase Annotation ```

Answer 7

1. The DNA has to be fragmented | 2. Clonal amplification (different PCR methods)

Answer 8

Slow and produces short sequence lengths

Answer 9

Illumina | Ion torrent

Answer 10

Oxford Nanopore Ion torrent Illumina

Answer 11

Reversible terminators

Answer 12

Don't have to move DNA around | Paired end sequencing

Answer 13

millions of dense clusters of dsDNA

Answer 14

1. Fragment DNA and attach to surface of flow cell using adaptors which contains a primer 2. Initiate bridge PCR with unlabelled NTPs 3. When PCR complete, millions of clusters are formed, add 4 labelled reversible terminators, primers and DNA pol to begin sequencing. 4. Excite clusters with a laser allowing fluorescents to be captured 5. Remove reversible terminators and repeat cycle by adding more ddNTPs 6. data aligned and compared to reference sequences

Answer 15

Exploits the fact that addition of a dNTP | to a DNA polymer releases an H+ ion

Answer 16

1. The input DNA is fragmented, Adaptors are added and one molecule is placed onto a bead. 2. molecules are amplified on the bead by emulsion PCR 3. Each bead is placed into a single well of a slide. The slide is flooded with a single species of dNTP, along with buffers and polymerase, one NTP at a time. 4. The pH is detected in each of the wells, as each H+ ion released will decrease the pH.

Answer 17

The lack of DNA or RNA amplification in template library preparation.

Answer 18

1. Avoid polymerase chain reaction-introduced error and amplification bias. 2. Real time measurements 3. Much higher throughput than first and second generation techniques. 4. Lower price per Mbp generated sequence 5. Longer reads

Answer 19

zero-mode waveguide wells- creates an illuminated observation volume that is small enough to observe only a single nucleotide being incorporated by the DNA polymerase

Answer 20

1. The platform uses a DNA polymerase anchored to the bottom surface of a ZMW. 2. Differentially labelled nucleotides enter the ZMW via diffusion and occupy the 'detection volume' . 3. During an incorporation event, the labelled nucleotide is 'held' within the detection volume by the polymerase for tens of milliseconds. 4. As each nucleotide is incorporated, the label located on the terminal phosphate is cleaved off and diffuses out of the ZMW

Answer 21

Very efficient: fewer expensive chemicals have to be used Very sensitive Long reads (10-15 Kbp)

Answer 22

Strengths: cheap, fast, simple, large reads, scalable, … | Problem: rather high error rate

Answer 23

measures the probability that a base is incorporated incorrectly (Higher Score indicates a smaller probability of error).

Answer 24

A method of quality encoding during genome assembly.

Answer 25

De novo= No scaffold sequence to guide alignment. | Read mapping=Reads are aligned to a reference sequence.

Answer 26

Greedy method- Incremental build. Builds using highest scoring overlap. Graph based: Pre-processes the data to produce a graph structure of pairwise overlap info.

Answer 27

de Bruijn- Split short reads into shorter uniform reads.

Answer 28

The length of the string

Answer 29

A genome assembler that use de Bruijn graph based algorithm. Takes short reads and removes errors to produce high quality contigs.

Answer 30

The N50 value informs researcher that 50% of the genome is assembled in contigs larger than the N50 value.

Answer 31

Attaching biological meaningful info to genome sequences.

Answer 32

Open reading frame- Part of the reading frame that contains no stop codons. UAA UGA UAG

Answer 33

AB initio method- Uses info from genomic sequence such as GC content and ORFs Database similarity - Uses public databases to identify sequences that are similar Combiners: Use both techniques

Answer 34

A genome map as preliminary data

Answer 35

1. Fragmentation of genome 2. Inserted into plasmid 3. Plasmid Cloned in e.coli 5. Investigate order of clones in genome using hybridisation, to give full genome map. 6. Take each clones and fragment DNA 7. Apply sequencing method 8. Form contigs 9. Merge contigs

Answer 36

1. initiated by the addition of unlabelled NTPs. 2. DNA amplification occurs and then DNA folds and form a bridge due to hybridisation of adapters. 3. Add polymerase and primers to make a complementary strand. 4. Increase the temp to break bridge connection leaving two ssDNA, the forward and reverse strand. 5. This form millions of clusters for illumina sequencing