Week 8 (Variants & SNP Chips)

Question 1

_____ _________ based on multiple metrics (need to be determined empirically) for variant filtering

Answer 1

hard filtering

Question 2

what are the two most important metrics in hard filtering?

Answer 2

• QualByDepth (QD)
• RMSMapping Quality (MQ)

Question 3

VCF

Answer 3

variant call format

Question 4

MQ (RMSMappingQuality) has a value of 40 associated with it. What does that mean?

Answer 4

it allows us to evaluate how good we think the gene mapped to the genome

Question 5

where would you find a lower MQ (mapping quality)?

Answer 5

in repetitive sequences because there are multiple places it could go

Question 6

sensitivity

Answer 6

identifying true positives

Question 7

specificity

Answer 7

identifying true negatives

Question 8

all variant callers produce errors. these errors can be classified as false positives and false negatives. when performing a genomic analysis, or any similar analysis for that matter, on has to balance sensitivity and specificity, what do the terms sensitivity and specificity mean in the context of variant calling?

Answer 8

sensitivity: trying to discover all the real variants
specificity: trying to limit the false positives that creep in when filters get too lenient

Question 9

if you call a variant where one doesn’t exist this is a false __________

Answer 9

positive

Question 10

if you fail to identify where a variant exists it is a false __________

Answer 10

negative

Question 11

what type of error is considered the worst?

Answer 11

Type 1 error (we don’t want to say something is true if it is not)

Question 12

Variant Quality Score Recalibration

Answer 12

does not actually recalibrate QUAL but creates a new score

Question 13

what is the purpose of the variant quality score recalibration?

Answer 13

the purpose of this new score is to enable variant filtering in a way that allows analysts to balance sensitivity and specificity

Question 14

sensitivity in variant quality score recalibration

Answer 14

trying to discover all the real variants

Question 15

specificity in variant quality score recalibration

Answer 15

trying to limit the false positives that creep in when filters get too lenient

Question 16

________ _______ ________ ___________ uses machine learning algorithms to learn from each dataset what the annotation profile of good variants vs bad variants

Answer 16

variant quality score recalibration

Question 17

VQSR

Answer 17

variant quality score recalibration

Question 18

key to VQSR is that you need a “________ _____” for training the model

Answer 18

truth set

Question 19

what is 100% sensitivity?

Answer 19

calling every difference a variant

Question 20

the recalibrated variant quality score provides a continuous estimate of the probability that each variant is true, allowing one to partition the call sets into quality ____________

Answer 20

tranches

Question 21

__________ are essentially slices of variants, ranked by VQSLOD

Answer 21

tranches

Question 22

high tranche

Answer 22

if you want more variants and are willing to accept false positives

Question 23

middle tranche

Answer 23

if you want to remove most false positives but are also willing to remove some true variants

Question 24

low tranche

Answer 24

if you only want highly accurate true variants with few false positives and willing to miss perhaps many true positives

Question 25

what are tranches?

Answer 25

slices in the variant quality scores, where to set the threshold to identify the amount of true positives and accept a number of false positives

Question 26

slices in the variant quality scores, where to set the threshold to identify the amount of true positives and accept a number of false positives

Answer 26

tranches

Question 27

what is a genotyping model and software that google has released?

Answer 27

Deep Variant

Question 28

what is the standard genotyping model and software used in humans?

Answer 28

Deep Variant

Question 29

WGS

Answer 29

whole genome sequence

Question 30

what is a whole genome sequence (WGS)?

Answer 30

the sequence library

Question 31

what is a SNP Chip used for?

Answer 31

to build a (relatively) low cost assay to genotype a large number of individuals

Question 32

sample size is statistical ________

Answer 32

power

Question 33

what is deep coverage?

Answer 33

30 x

Question 34

approximately how much does it cost to run a whole genome sequencing on a mammalian genome at 30x coverage?

Answer 34

$1000

Question 35

what is the difference between WGS and SNP chips related to variants?

Answer 35

- WGS captures "all" variation - SNP chips have lower number of variants but also a lower cost per sample

Question 36

_______ is the largest genotype provider in the world

Answer 36

Neogen

Question 37

what was the purpose of in-silico digest of reference genome with multiple restriction enzymes?

Answer 37

every time a sequence was seen it would cut it, from there you could compile the amount of reads you had from each segment and the repetitive elements (that you are not interested in) could be found because they had the most sections cut out

Question 38

what is illuminated infinium chemistry?

Answer 38

small beads have unique barcodes, for each SNP 50 mer oligos flank it called probes, attach these SNP specific probes to the beads and then create a chip that has microwells for each of the beads to sit in, then deposit the beads on the chip to produce an array

Question 39

what is the basics of the beads used in illumina indium chemistry?

Answer 39

the small beads have oligos hanging off of them that correspond to the section of the sequence that you want

Question 40

for each SNP, synthesize a ____ mer oligo that flanks the SNP (probe)

Answer 40

50

Question 41

what is a probe?

Answer 41

50 mer oligo that flanks the SNP

Question 42

_____ base probe

Answer 42

50

Question 43

infinitum I = ____ probe(s)

Answer 43

2

Question 44

infinitum II = ____ probe(s)

Answer 44

1

Question 45

what color bead is G and C in illumina infinitum chemistry?

Answer 45

green

Question 46

what color bead is A and T in illumina infinitum chemistry?

Answer 46

red

Question 47

G/C and A/T = ____________

Answer 47

infinium I

Question 48

in illumina infinium chemistry, what happens if your variant is As AND Ts? How do you solve this?

Answer 48

it would all show up as red, so you would need to use two probes

Question 49

why do we cluster SNPs?

Answer 49

so we can determine genotype

Question 50

is this a well clustered SNP or a poorly clustered SNP?

Answer 50

well clustered SNP

Question 51

are these example of a well clustered SNP or a poorly clustered SNP?

Answer 51

poorly clustered SNP

Question 52

what are these clustered SNPs an example of?

Answer 52

improperly clustered SNPs, the automated system just got it wrong, so you should manually fix it

Question 53

SNP chips are really accurate but things can go wrong. remember this when making decisions based on chip genotypes for any single SNP specifically. Why?

Answer 53

it depends on what error rate you are comfortable with, your willingness to be wrong

Question 54

call rate per SNP

Answer 54

best indicator of genotype quality

Question 55

call rate per individual

Answer 55

best indicator sample of DNA quality

Question 56

2 key metrics for looking for errors:

Answer 56

1. call rate per SNP 2. call rate per individual

Question 57

why might for an individual have a low call rate?

Answer 57

poor DNA (for example taking from a live cow vs a cow that has been dead for 1000 years)

Question 58

what does it mean to impute?

Answer 58

taking missing data from data that you have already observed and filling in the gaps

Question 59

what do the signs on the right symbolize?

Answer 59

whole genome sequence: it is high density and all the variants have been found

Question 60

what do the signs on the left symbolize?

Answer 60

SNP Chip: low density