Week 7 (Variant Calling)

Question 1

after aligning the genome, we will want to identify ___________

Answer 1

variants

Question 2

what is a variant?

Answer 2

differences between our sample and the reference

Question 3

what are the types of variants we are exploring in this class?

Answer 3

• SNPs
• Indels

Question 4

SNPs

Answer 4

single nucleotide polymorphisms

Question 5

Indels

Answer 5

insertions/deletions (small <50 bp)

Question 6

unaligned sequence data file formats

Answer 6

• FASTA
• FASTQ

Question 7

Aligned sequence data file formats

Answer 7

• SAM
• BAM
• BAI
• CRAM

Question 8

SAM

Answer 8

sequence alignment map

Question 9

BAM

Answer 9

binary (compressed) version of SAM

Question 10

BAI

Answer 10

index for BAM

Question 11

variant calls (SNPs and Indels)

Answer 11

• VCF
• BCF

Question 12

VCF

Answer 12

variant call format

Question 13

BCF

Answer 13

binary of vcf (compressed version)

Question 14

mandatory fields of SAM

Answer 14

• what [4]
• where [5]
• how good (or bad) [2]

Question 15

aligners ouput a _____ file that is then compressed to a ______ file

Answer 15

SAM; BAM

Question 16

what is MAPQ?

Answer 16

in the SAM file, MAPQ will tell you how good of a job we do mapping the read to the reference. the lower the map qualities the worse we did at mapping.

Question 17

what parts of the genome often have low MAPQ?

Answer 17

repetitive regions and simple regions

Question 18

do we prefer systematic errors or random errors? why?

Answer 18

• we prefer random error
• random error occurs randomly and can be overcome. systematic errors are difficult to find and can mess up all of the data.

Question 19

________ errors are preferred. ________ errors cause issues with downstream analysis.

Answer 19

random; systematic

Question 20

what happens if there are errors that occur during PCR, what happens to your data?

Answer 20

the error is transferred all the way down your analysis

Question 21

two types of duplicate reads

Answer 21

• PCR
• optical

Question 22

what is optical duplicate read?

Answer 22

more prevalent on patterned flow cells (due to imaging on older flow cells and exAmp chemistry on patterned flow cells)

Question 23

how are duplicates identified?

Answer 23

based on the starting positions of the read

Question 24

what is a downside to using the patterned flow cell?

Answer 24

you end up with far more optical duplicates

Question 25

BQSR

Answer 25

base quality score recalibration

Question 26

almost all sequence analyses use the _____ _______

Answer 26

base quality

Question 27

quality values reported by sequencer are __________ or error rate

Answer 27

estimates

Question 28

BQSR uses a set of known variant positions (dbSNP) and considers all other variants from sequence data as _________

Answer 28

errors

Question 29

how does base quality score recalibration work?

Answer 29

uses a set of known positions in the genome that are known to be variable, we expect an individual may be different at that position, so we will only look at where our data is different from other parts of the reference, if our individual is different it may be due to a sequencing error

Question 30

when observing the reported quality vs the empirical quality (expected quality) of the data, we will just adjust the data to fit the distribution. For which sequencer may we not have to recalibrate the data?

Answer 30

element because it is so accurate

Question 31

when comparing the reference genome to the DNA of the animal that the reference was made of, what would the expectation be?

Answer 31

there would be none to very little difference (maybe show some heterozygosity)

Question 32

what are two common variant callers?

Answer 32

- GATK HaplotypeCaller - DeepVariant (more common)

Question 33

two approaches to variant calling

Answer 33

- single sample - joint

Question 34

_____ calling is always better

Answer 34

joint

Question 35

why is joint calling better than single sample calling?

Answer 35

It improves variant detection by using data from multiple samples to enhance sensitivity, especially for rare variants. This approach increases accuracy, reduces false positives, and strengthens analysis by leveraging collective sample information, making it particularly useful for large studies.

Question 36

key benefits to HaplotypeCaller

Answer 36

- call SNPs and indels simultaneously - local de-novo assembly - output gVCF for cohort (joint) calling

Question 37

4 stages for HaplotypeCaller

Answer 37

1. define active regions 2. local assembly to build haplotypes 3. estimate likelihoods of the haplotypes given the data 4. assign sample genotypes

Question 38

HaplotypeCaller: define active regions

Answer 38

regions of the genome based not eh presence of significant evidence for variation

Question 39

as an example, if a kmer was 7 long, how many of the first kmer are TATGAAA vs TATGCAA?

Answer 39

- 4 Kmers with TATGAAA - 4 Kmers with TATGCAA

Question 40

HaplotypeCaller: determine haplotypes by local assembly of the active region

Answer 40

for each region, build a de bruijn-like graph to reassemble the active region and then identify the active haplotypes present and realign using the smith waterman algorithm

Question 41

why would you use the smith waterman algorithm to identify the active hapltypes?

Answer 41

it is guaranteed to find the optimal local alignment

Question 42

haplotype

Answer 42

a group of alleles (different versions of a gene) located close together on a single chromosome that are inherited together from one parent, essentially a set of DNA variations that tend to be passed down as a unit due to their proximity on the chromosome

Question 43

key benefits of HaplotypeCaller

Answer 43

- call SNPs and indels simultaneously - local de-novo assembly - output of gVCF for cohort (joint) calling

Question 44

4 stages of HaplotypeCaller

Answer 44

1. define active regions 2. local assembly to build haplotypes 3. estimate likelihoods of the haplotypes given the data 4. assign sample genotypes

Question 45

gVCF ouput file allows _____ calling

Answer 45

cohort

Question 46

VCF

Answer 46

variant call format

Question 47

what are the 8 mandatory fields in a VCF file?

Answer 47

- what: ID/REF/ALT/INFO - where: CHROM/POS - how good: QUAL/FILTER

Question 48

hard filtering

Answer 48

based on multiple metrics, used to filter variants

Question 49

sensitivity

Answer 49

(true positive rate) measures the proportion of positives that are correctly identifies (the proportion of those who are affected and who were correctly identified as affected)

Question 50

specificity

Answer 50

(true negative rate) measures the proportion of negatives that are correctly identified (the proportion that are not affected and identified as not being affected)

Question 51

what is a type 1 error?

Answer 51

false positive

Question 52

what is a type 2 error?

Answer 52

false negative

Question 53

VQSLOD

Answer 53

variant quality score log-ODds

Question 54

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

Answer 54

- sensitivity - specificity

Question 55

variant filtering: sensitivity

Answer 55

trying to discover all the real variants

Question 56

variant filtering: specificity

Answer 56

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

Question 57

variant quality score recalibration uses machine learning algorithms to learn from each dataset what is the annotation profile of ______ variants vs _______ variants

Answer 57

good variants vs bad variants