Week 6 (QC and Alignment)

Question 1

the ability to resolve a repetitive structure is dependent on the __________ of the molecules in your library

Answer 1

length

Question 2

Sanger sequencing is ~_______bp accurate

Answer 2

1000

Question 3

short read sequencers

Answer 3

illumina and element

Question 4

short read sequencers use <____ bp and have a _______ error rate

Answer 4

<150bp; low error rate («1%)

Question 5

long read sequencers

Answer 5

• PacBio
• Oxford Nanopore

Question 6

long read sequencers have a lower number of reads but much longer being ____ to _____ kb, with a _______ error rate of ~____%

Answer 6

10’s to 100’s kb; higher error rate of ~1%

Question 7

what are the standard file formats?

Answer 7

• FASTA
• FASTQ

Question 8

FASTA has ___ parts

Answer 8

2

Question 9

what are the parts of FASTA

Answer 9

1. > sequence name (always has >)
2. sequence

Question 10

FASTQ has ___ parts

Answer 10

4

Question 11

what are the parts of FASTQ

Answer 11

1. @ sequence name (and other info)
2. sequence
3. • (sometimes other info)
4. quality value

Question 12

when is FASTA used?

Answer 12

when per base quality is not needed

Question 13

what does FASTA present?

Answer 13

presents only the sequence itself

Question 14

the FASTA sequence name always starts with ______ symbol

Answer 14

>

Question 15

when is FASTQ used?

Answer 15

when per base quality is needed

Question 16

what does FASTQ present?

Answer 16

presents the sequence and the estimated base quality

Question 17

FASTQ sequence name always starts with ____ symbol

Answer 17

@

Question 18

when reading the sequence in FASTQ, what does the letter “N” symbolize?

Answer 18

any of the 4 nucleotides, it did not know which nucleotide it was, so the more N’s the worse the quality

Question 19

the _______ the Q value the more accurate the sequence is

Answer 19

higher

Question 20

Quality scores increase by a factor of _____

Answer 20

10

Question 21

Qphred equation

Answer 21

Qphred = -10log10P(error)

Question 22

At any given position in a sequence, the base present is either A/C/T/G but we cannot _________ observe the base. The base that is produced from a DNA sequencer is an observation based on some biochemical/physical property that has an ________.

Answer 22

directly; error

Question 23

Q20 is _____ times more accurate then Q10

Answer 23

10

Question 24

when using fluorescence in illumina, we notice a change in color distribution with each cycle. How does this affect our accuracy?

Answer 24

clear signal intensity decreases as you do more cycles. this occurs because their may have been failure to cleave previous fluors on nucleotides

Question 25

Fred scale quality values of ___ or less (__, ___, ___, ___)

Answer 25

40 or less (30, 20, 10)

Question 26

in the program, the phred scale quality values are a ______ character, saving a lot of space

Answer 26

single

Question 27

what is a error that can occur in element? does this happen often?

Answer 27

single base error, however this does not happen very often

Question 28

what is FASTQC?

Answer 28

- QC = quality control - one of the many software tools to evaluate quality

Question 29

FASTQC does not actually do any filtering it....?

Answer 29

provides summary metics and visuals

Question 30

In Base Quality - is this good data or poor data?

Answer 30

good data

Question 31

In Base Quality - is this good data or poor data?

Answer 31

poor data

Question 32

in per tile sequence quality, which os the good data and which is the poor data? how do you know?

Answer 32

good data on the left and poor data on the right. on the poor data there must have been a problem between cycle 5 and 15 in this location in the flow cell because there are lines shown.

Question 33

in these per sequence quality scores, which one has good data and which has poor data? how do you know?

Answer 33

good data on the left has a high sequence quality almost to the end of the reads. the poor data is on the right with sequence quality that begins to decrease earlier in the reads.

Question 34

in the per base sequence content, which has good data and which has poor data? how do you know?

Answer 34

good data on the left because it has a uniform base composition to the end of the reads. the poor data on the right's base composition diverges indicating problems.

Question 35

error probability: Q10

Answer 35

0.1 (1 in 10)

Question 36

error probability: Q20

Answer 36

0.01 (1 in 100)

Question 37

error probability: Q30

Answer 37

0.001 (1 in 1000)

Question 38

error probability: Q40

Answer 38

0.0001 (1 in 10000)

Question 39

k-mer

Answer 39

a substring of a fixed length that appears in a biological sequence

Question 40

4^(kmer length)

Answer 40

number of possible nucleotides

Question 41

are K-mers odd or even integers?

Answer 41

use odd integers

Question 42

sequence alignment

Answer 42

a string matching problem, using a reference genome to match your sequences

Question 43

two main algorithms for alignment | (of short read sequencing data)

Answer 43

- smith-waterman - burrow-wheeler transform

Question 44

smith-waterman

Answer 44

guaranteed to find the optimal local alignment with respect to the coring matrices used

Question 45

what is smith-waterman best used for?

Answer 45

good for aligning "Sanger" length data and it is too slow for high throughput data

Question 46

burrow-wheeler transform

Answer 46

creates a suffix array of smaller k-mers, index the reference genome, match the seed of the read to the reference and extend seed to full alignment

Question 47

what are the most used aligners?

Answer 47

BWA (WGS) and STAR (RNA)

Question 48

what two algorithms for alignment are best used in short read sequencing data?

Answer 48

- smith-waterman - burrow-wheeler transform

Question 49

what algorithm is used for long read? | (in alignment)

Answer 49

minimap2

Question 50

two main sequence file formats

Answer 50

FASTA and FASTQ

Question 51

high throughput sequence data needs _____

Answer 51

QC

Question 52

what softwatr tool provides summary and visualization to evaluate quality

Answer 52

FastQC

Question 53

sequence alignment

Answer 53

- smith-waterman - burrow-wheeler transform (BWA and STAR)

Question 54

what does "depth" mean when sequencing?

Answer 54

the amount of times it has been sequenced

Question 55

what is the ability to resolve a repetitive structure dependent on?

Answer 55

the length of the molecules in your library

Question 56

when analyzing a pileup, you notice that one of the lines has equal parts A and C. What does this mean? Is this an error in the sequence?

Answer 56

this is most likely not an error, this is showing that on the chromosome from one parent you got an A and the other you had a C, this is representing both chromosomes (heterozygous)