Genome assembly

Question 1

Why is genome assembly necessary?

Answer 1

no seq technology can produce long enough reads

it is the rate limiting step in genomics

Question 2

De Novo vs Re-Sequencing

Answer 2

De novo: determination of a full-genome sequence, NO known reference sequence. Needs a lot of sequence coverage and computing power.

Re-sequencing: a reference genome sequence is known. The assembly process is replaced by mapping the raw sequence reads onto the reference genome. Less sequence coverage and computing power needed.

Question 3

what is resequencing good and bad at detecting?

Answer 3

Good - SNPs

Bad - limited in the detection of structural rearrangements (insertions, deletions, inversions).

Question 4

How to calculate coverage

Answer 4

bases needed to assemble a sequence/bases in the sequence

Question 5

How do assembly algorithms work and what are 2 difficulties?

Answer 5

search for overlaps between sequence reads

• data volume requires high comp power
• repeat regions which are larger than the read

Question 6

relationship btw seq coverage and probability of detection

Answer 6

sigmoidal (S curve)

Question 7

what is a contig?

Answer 7

partial assembly of data from overlapping fragments into a contiguous region of sequences. The order of the contigs is NOT known.

Question 8

why are repetitive regions problematic in assembly

Answer 8

regions of receptive seqs can mean that contorts cannot join up.

Question 9

describe paired end sequencing

Answer 9

• create library from sample DNA.
• isolate fragments which are about 800bp long.
• sequence 250bp from each end of the fragments using illumina.
• now, the sequence of both ends is known and we know the ends are about 800 bp apart. in-between is unknown.
• can map fragments to genome.

Question 10

when is paired end seq particularly useful?

Answer 10

sequencing of fragments that contain short repeat regions, because paired end reads have relatively small inserts.

Question 11

what is the difference btw paired end fragments and mate pair fragments?

Answer 11

mate pair frags have larger inserts (3kb-15kb), paired end has about 800bp.
Mate pair enables coverage of regions with large structural rearrangements.

Question 12

example of 2 long read seq tehcs

Answer 12

PacBio and ox nanopore

Question 13

how can long read seq tacos be used

Answer 13

can make v long reads but high error rates
so, make initial assembly with long read, then additional illumina short read seq for error correction.
New genome assemblers directly incorporate PacBio and Illumina.

Question 14

What is Bionano

Answer 14

optical mapping system

Question 15

How does optical mapping work?

Answer 15

• non-destructive restriction map (no fragmenting)
• single-stranded nick is inserted into double-stranded DNA at positions of a seven-base recognition site. (uses 7bp cutter enzyme) cute every 4^7 bases.
• nick is ligated by insertion of a fluorescent marker, dispersing visible signals along the genome

Question 16

How is optical mapping used?

Answer 16

By visualizing large-scale segments of the genome, and comparing with a reference, it is possible to detect translocations, repeats and deletions.
In conjunction with sequencing of fragments it is possible to apply the map to assemble a complete genome sequence.

Question 17

BionanoIrys system

Answer 17

used to check contig placement.

can detect large structural misassemblies

Question 18

what is a scaffold?

Answer 18

ordered stretch of contigs

contains sequence gaps NNNNN

Question 19

What approaches are used to build scaffolds?

Answer 19

using approaches such as optical maps
chromosome contact maps
10X genomics

Question 20

which machine does 10X genomics?

Answer 20

10X genomics chromium

Question 21

How does 10x genomics work?

Answer 21

• partitions large DNA molecules (50-100 kb) into small droplets. Each droplet is assigned a unique barcode.
• each molecule sequenced using illumina and each read can be certainly assigned to a large DNA molecule.
• works with tiny amount of DNA 1ng, so good for single cell analysis.

Question 22

2 approaches to whole genome seq projects

Answer 22

1. Hierarchial approach - whole genome fragmented and cloned into BACs. order of fragments established before seq
2. whole genome shotgun method - large numbers of smaller fragments. harder assmebly

Question 23

how much DNA can a BAC carry

Answer 23

100-200kbp

can store foreign DNA 10x larger than normal plasmids

Question 24

BAC to BAC

Answer 24

DNA is cut into fragments of about 150 kb. Fragments are cloned into BACs.
do enough to get good coverage
- BAC clones are ordered by fingerprinting, e.g. based on overlapping restriction fragment size patterns.
- individual BAC clones are sequenced, sequences of each BAC clone assembled independently.
- since order is known, can infer order of sequence.

Question 25

advantage of BAC to BAC

Answer 25

1. dramatically reduced the size and complexity of the 2.genome piece that needs to be considered.
2. Assemble algorithm only has to deal with a frag of 150kb.
3. repeat problem reduced. even if highly repetitive element occurs across genome, unlikely it will appear in the same BAC clone.

Question 26

Whole-genome shotgun approach differs from BAC to BAC?

Answer 26

Different to BAC to BAC - order of genome fragments is not determined prior to sequencing.
much higher computing power needed
bypass lengthy fingerprinting of thousands of BAC clones

Question 27

Which is used more now? whole genome shotgun approach or BAC to BAC

Answer 27

Shotgun now due to novel seq techniques and scaffolding approaches

usually combines different technologies for seq and also for scaffolding and error correction.

Question 28

How much coverage is needed for a whole-genome shotgun approach in order to receive a decent quality?

Answer 28

Lander and Waterman:

c=NL/G
= no. reads*read length/genome length

accepted coverage = 100

Question 29

What has seriously confounded whole-shotgun genome assemblies

Answer 29

presence of repetitive elements

not considered in the formulas for coverage

Question 30

barley genome

Size and how was it sequenced?

Answer 30

genome released in 2017
4.8Gb (largest sequenced at high quality)
Hieracrchal BAC to BAC approach used
87,075 BAC clones were sequenced, mainly using Illumina paired-end and mate-pair technology. Each BAC clone was assembled individually.
Scaffolding done using optical map and chromosome conformation capture sequencing (Hi-C).

Question 31

How to measure genome quality?

Answer 31

1. length of the total assembly should be close to the estimated genome size
   Barley - 4.79Gb compared to 4.8Gb.
2. low no. contigs. N50 should be high
3. scaffold N50 should be high

Question 32

what is N50?

Answer 32

50% of the genome is formed of contigs or scaffolds of this size or larger.

Question 33

Annotated coding sequence (in assembly annotation statistics)

Answer 33

if it is low, makes whole genome approach challenging

Question 34

benchmarks for reference genomes, decided by Vertebrate genome project 2018

Answer 34

N50 size contig: >1 Mb

N50 size scaffold: >10 Mb

Sequence error frequency: < 1 in 10,000 bases (phred score = 40)

Structural variants be confirmed by multiple technologies

90% of the sequence be assigned to chromosomes.