Weeks 5-6: Genome Assembly; BLAST/FASTA

Question 1

The IHGSC used a _____ approach to sequence the genome

Answer 1

hierarchical

Question 2

Celera used ______ to sequence the genome

Answer 2

whole genome shotgun

Question 3

Pseudogene

Answer 3

DNA sequence resembling a gene but mutated into an inactive form over the course of evolution.
Often lacks introns and other essential DNA sequences necessary for function. Pseudogenes do not result in functional proteins, but may have regulatory effects

Question 4

True or false:

98% of the genome doesn’t encode proteins

Answer 4

True. Only 2% of the genome encodes proteins.

The other 98% encodes small RNAs that regulate gene expression

Question 5

Output of Sanger sequencing

Answer 5

Single sequence ranging from 500-1000 bp

Question 6

Output of Next-Gen Sequencing

Answer 6

Groups sequences ranging from 25-500 bp

Question 7

De novo assembly

Answer 7

Reconstruction of contiguous sequences without making use of any reference sequence.
Reads are partitioned into k-mers (substrings of the read sequence of length k)-form the nodes of the graph (network) and are linked when sharing a k-1 mer.

Question 8

Genome annotation

Answer 8

Computationally expensive process attaching biologically relevant information to genome sequence data

Question 9

Pre-Assembly Steps

Answer 9

FastQC: To check the quality of the sequencing data, overall GC content, repeat abundance, the proportion of duplicated reads.
Trim sequences: adapter trimming (cutadapt), trim reads based on quality (sickle).
Remove contaminant sequences such as DNA from the PhiX phage: use a short read aligner (such as Burrows-Wheeler Aligner)
Demultiplex reads: Galaxy

Question 10

True or false:

Gel electrophoresis is required for NGS

Answer 10

False. No gel electrophoresis needed.

Question 11

Smith-Waterman Search

Answer 11

Perform dynamic programming between query and each sequence in the collection.
Accurate - guaranteed to report the highest scoring alignments
Slow - searching a 52,000,000,000 basepair collection (entire GenBank database) takes around 3 days on a modern workstation

Question 12

FASTA

Answer 12

First heuristic search algorithm
~5x faster than Smith-Waterman
Four stage search process - first stage based on algorithm of Wilbur and Lipman for finding exact matches of length n between query and collection sequences

Question 13

Wilbur-Lipman Approach

Answer 13

• Ignore indel events
• Extract intervals (fixed-length overlapping subsequences from the first sequence of length n)
• Store intervals in fast search structure
• For each interval in the second sequence, search for it in the hash table

Question 14

FASTA steps

Answer 14

Step 1: Identify regions shared by the two sequences of length n = 1 (using the Wilbur-Lipman method)
Step 2: Rescan the top-ten regions, and rescore using a scoring matrix (protein only)
Step 3: Check to see if initial regions can be joined to form rough alignment with gaps
Step 4: Perform banded Smith-Waterman location alignment centred around all regions that score greater than a threshold

Question 15

BLAST

Answer 15

~50x faster than Smith-Waterman, 10x faster than FASTA but not 100% accurate
Stage 1: BLAST searches for hits (matches of length W between query and subject). Location of each hit is passed to stage 2.
Stage 2: BLAST performs an ungapped alignment of region surrounding each hit. High-scoring ungapped alignments (where score > T) are passed to stage 3
Stages 3 and 4: BLAST performs a gapped alignment of region surrounding each high-scoring ungapped alignment
High-scoring alignments are displayed to the user

Question 16

Difference between BLAST protein and nucleotide searches

Answer 16

Blast Protein Search - Two-hits on the same diagonal (instead of just one) are required to trigger an ungapped alignment.

Question 17

Why are index-based approaches not suitable for searching large collections?

Answer 17

Because the index ends up being much larger than the data itself

Question 18

True or false:

Two proteins that are related in recent evolutionary terms will usually share sequence and structural similarity

Answer 18

True

Question 19

PSI-BLAST

Answer 19

Used to detect distantly related homologues not detected by BLASTP.
Several rounds (iterations) of BLAST are run. Between each round, a Position-Specific Scoring Matrix (PSSM) is constructed, used for the subsequent iteration.

Question 20

PSI-BLAST

Answer 20

Used to detect distantly related homologues not detected by BLASTP.
Several rounds (iterations) of BLAST are run. Between each round, a Position-Specific Scoring Matrix (PSSM) is constructed, used for the subsequent iteration. If new matches are found, another matrix is constructed. If no new matches found, hits with a E<1x10^-6 are recorded.

Question 21

Significance threshold for PSI-BLAST

Answer 21

Experimental tests of PSI-BLAST using default parameters have determined that proteins identified in the first 20 iterations with expect scores <1x10^-6 are most likely real

Question 22

Define: Domain

Answer 22

contiguous stretch of amino acids “that look as though they should have independent stability”.

Question 23

Decreasing the e-value threshold reduces the likelihood of _______ but decreases ________.

Answer 23

false positives; sensitivity

Question 24

False positives in PSI-BLAST searches are:

Answer 24

High-scoring alignments that are not in fact related to the query

Question 25

What is required to trigger an ungapped alignment in BLASTn?

Answer 25

One hit on the same diagonal

Question 26

What is required to trigger an ungapped alignment in BLASTP?

Answer 26

Two hits on the same diagonal

Question 27

Stages of BLAST

Answer 27

1. BLAST searches for hits
2. BLAST performs ungapped alignment. High scoring ungapped alignments are passed to stage 3.
3. BLAST performs gapped alignment of region surrounding each high-scoring alignment.
4. High scoring alignments are presented to the user.

Question 28

What types of reads are generated using shotgun sequencing?

Answer 28

whole-genome shotgun reads

Question 29

What types of reads are generated using hierarchical sequencing?

Answer 29

BAC shotgun reads

Question 30

Advantages of MSA over pairwise alignments

Answer 30

More information than pairwise alignment
Can create phylogenetic trees
Can identify conserved regions

Question 31

ClustalW steps

Answer 31

1. Begins with pairwise alignment and scoring all the pairs
2. Builds phylogenetic tree
3. Most closely related sequences are aligned and form a consensus using dynamic programming. The next closest related sequences are then aligned and form a consensus and so forth.

Question 32

Iterative search

Answer 32

1. Search database with query sequence
2. Construct multiple alignment from high-scoring aligned sequences
3. Construct a profile using the multiple alignment
4. Search database with profile. Repeat.

Question 33

E-value

Answer 33

The probability that the sequence is similar to the probe sequence purely by chance

Question 34

Which profile does PSI-BLAST use?

Answer 34

Position-specific score matrices (PSSMs)

Question 35

What profile does SAM use?

Answer 35

Hidden Markov Models (HMMs)

Question 36

Examples of iterative searches

Answer 36

PSI-BLAST and SAM