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Genomics > Week 5 (Long Read and Element) > Flashcards

Week 5 (Long Read and Element) Flashcards

1
Q

ability to resolve a _________ structure is dependent on the length of the molecules in your library

A

repetitive

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2
Q

segmental duplications

A

“low copy repeats” blocks that range from 1 to 400 kb in length, occur at more than one site within the genome, and typically share a high level (>90%) of the sequence identity.

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3
Q

segmental duplications make up about ____% of the human genome

A

5

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4
Q

long read technology

A
  • Oxford nanopore (ONT) (protein nanopores)
  • Pacific BioSciences - PacBio (SMRT)
  • proximity ligation (assembly)
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5
Q

ONT

A

Oxford nanopore (protein nanopores)

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6
Q

SMRT

A

single molecule real time sequencing

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7
Q

__________ is a heptameric protein pore with an inner diameter of a few nanometers

A

a-hemolysin

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8
Q

the diameter of a-hemolysin is the same scale as many single molecule, including DNA. Why?

A

so that DNA can be extruded from the membrane

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9
Q

ONT (protein nanopore) can be used real time in the field. 10-20 Gb are read in less than _______

A

24 hours (standard is 72 hours)

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10
Q

where is a-hemolysin derived from?

A

it was discovered in staph, the pathogenic organism uses this protein ore to penetrate cells in the body

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11
Q

how is DNA extruded from the cell using protein nanopore?

A

the pore is in the membrane, there is a tether that holds the DNA on the pore and a motor protein allows the DNA to move through the pose

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12
Q

How do we read the bases as the exit the protein pore?

A

as the DNA goes through the pore, each base has its own structure that will disrupt the charge in a base specific way (ion current), so we can estimate what is coming out based on the change in charge

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13
Q

using protein pores, _____ bases are read per second

A

400

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14
Q

long read sequencers are important for resolving ________ sequences

A

repeat

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15
Q

____ Mb is the largest long read that has been read (the largest read is the largest chromosome)

A

4.2

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16
Q

selective sequencing

A

the protein nanopore is able to chose only the sequences that we are interested in, it will reject and eject the molecule if it has seen it already and then restart with a new sequence

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17
Q

why is selective sequencing a really great tool?

A

it will save time and resources

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18
Q

what can protein nanopores read in one read?

A
  • bases sequenced
  • bases inserted
  • bases deleted
  • SNVs
  • CpG methylations
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19
Q

centromeres are found in the ________ of the chromosome

A

middle

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20
Q

telomere are found on the _____ of the chromosome

A

end

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21
Q

what is the difference between illumina sequencing and ONT (protein pore)’s average read length?

A
  • illumina = 150 bp
  • ONT = 33-35 kb
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22
Q

what is a major benefit of ONT (protein nanopore)?

A

read length

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23
Q

what is the average read length of ONT (protein nanopore)?

A

33-35 kb

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24
Q

in ONT, about _____ bp/sec

A

400

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25
what type of error occurs in ONT (protein nanopore)? what is the accuracy?
- homopolymers - accuracy: >99%
26
homopolymers
types of repeats
27
SINEs: _____ bp LINES: _____ bp
- SINEs: 500 bp - LINES: 5,000 bp
28
What type of technology do LINEs use to get read, otherwise they will be mostly inaccurate?
LINEs will be read by long reads, short reads would mostly be inaccurate
29
ONT resolves the problem of ___________ __________ while illumina does not due to short reading
segmental duplications
30
nano-wells called zero-mode wave guides (ZMWs)
detecting wavelength, physically restricting the light when excited with a laser
31
where is the fluor in PacBio to read the sequence? what is its formal name?
on the phosphate of the nucleotide (phospholinked nucleotide)
32
the mean read lengths of pacbio are >_____ kb
20
33
what makes pacbio really accurate?
CCS / high fidelity system, it is able to go over the strand multiple times
34
would you rather have systematic error or random error?
random error is better than systematic because you can overcome random error with depth of sequencing (rerunning it) than systematic error that will not be overcome
35
what is a Q value?
probability of error per base
36
PacBio reads ~____bp/sec
10
37
what are common errors in PacBio?
homopolymers and indels
38
what is a homopolymer?
same sequence repeated over and over again
39
what is an indel?
(insertions and deletions) - a genetic mutation that occurs when one or more DNA bases are inserted or deleted from a genome
40
what is PacBio's accuracy?
>99%
41
what is the common error type in illumina? Why?
base substitution because it sequences one nucleotide at a time
42
what is the common error type in ONT? Why?
homopolymers and indels because it is working so fast that it isn't catching all of the sequence as it records the charge
43
what is the common error type in PacBio? Why?
homopolymers and indels because it is working so fast that isn't catching all of them
44
what is "proximity ligation: Hi-C"?
it is a library preparation step, genome scaffolding
45
when a linear line is coiled up, is it more likely to be closer to something a couple Kbs away or several mb away?
it will be closer to something a couple kb away (the closer in linear space, the closer in 3D space it should be)
46
when DNA is compacted into chromatin (a 3D structure), the DNA that is close together is ______________, trapping sequence interactions across the entire genome and between different chromosomes
cross linked
47
what does cross linking do?
cross linking traps sequence interaction across the entire genome and between different chromosomes
48
crossliked DNA is fragmented with ____________
endonucleases
49
what is proximity ligation?
after crosslinks are fragmented, they are then biotenelated and ligated creating chimeric junctions between adjacent sequences
50
what is proximity ligation used for?
to assign context to chromosomes and order and orient them along chromosome scale scaffolds
51
_______ is currently the long read technology of choice
PacBio
52
PacBio increases read lengths and increases throughput = ___________ cost per genome
decreasing
53
ONT has extremely long reads but relatively few making it ___________
expensive
54
which technology has no upper limit on the size of template that can be sequenced giving it huge potential?
Oxford nanopore (ONT)
55
what is the purpose of HiC?
order and orient contigs
56
Element is a competing ________ read sequencing
short
57
in element, an ______ is a dye-labeled polymer with multiple nucleotide arms carrying the same nucleotide base
avidite
58
how are the bases detected in element sequencing?
florescent signals in 4 channels correlate with A, T, C, or G avidities.
59
steps of element sequencing:
1. bind avidite 2. wash away unbound avidites 3. bases are detected 4. remove avidite 5. step and block 6. remove blocks 7. repeat
60
element is extremely good per base accuracy and does well with ____________. This is a great method for reducing error.
homopolymers

Genomics (17 decks)

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