bioinformatics exam 2

lec 8 to 15

BLAST

basic local alignment search tool

what is read mapping?

process of determining where a short DNA sequence originated within a reference genome

mapping hundreds of millions of reads back to the reference genome is what?

CPU and RAM intensive, causing to be slow

most mappers allow how many mismatches within the first what bp?

2 mismatches within the first 30bp, slower when allowing indels

indels

insertion or deletion of one or more nucleotides

what is a mismatch?

a nucleotide difference between a sequencing read and the reference sequence

why does allowing indels slow down read mapping?

mapper must evaluate additional possible alignments that include gaps

global alignment

align sequences across their entire lengths

local alignment

identifies the most similar regions within two sequences

semi global alignment

aligns sequences while allowing certain terminal gaps without penalties

what alignment does BLAST focus on

local alignment

hash based mapping

step 1: hash/index the genome
step 2: use the index to map (find alignment location) reads

what is kmer index used for?

quickly find candidate alignment locations in genome

what does it mean to index the genome?

recording where short DNA sequences occur within a larger reference sequence

what are kmers?

nucelotide sequence of length k

length k

represents how many nucleotides are included in each short sequence

What are two possible causes of a mismatch between a read and its reference genome?

A sequencing error or a genetic variant

Why is more rigorous downstream alignment needed?

To evaluate candidate alignment locations while allowing mismatches and indels

Does a sequencing read need to match the reference perfectly to be mapped?

No. Reads containing mismatches or indels may still be mapped using appropriate alignment methods

smith waterman

dynamic programming for local sequencing alignment

blast is a ___________ form of smith waterman because it is _____ and _______.

simplified, fast, accessible

heuristic

computational strategy that reduces the amount of searching required to find useful results

how does BLAST save time

by restricting the search by scanning database sequences for likely matches before performing more rigorous alignments

what does BLAST sacrifices

sensitivity in exchange for speed

sensitivity

describes an alignment method's ability to detect relevant sequence similarities

more sensitive method

ess likely to overlook a genuine alignment.

Does BLAST always find the optimal alignment?

no

True or False: BLAST is guaranteed to identify the optimal local alignment between two sequences.

FALSE

BLAST e-value

expectation value; The number of alignments expected to occur by chance with equivalent or better scores

Does a lower or higher E-value indicate greater statistical significance?

lower

What does an E-value of 0.005 indicate according to the lecture?

Approximately one such chance alignment is expected across 200 comparable random searches.

What E-value does your professor identify as a commonly used threshold for statistically significant matches?

0.005

Which is more statistically significant: E = 0.001 or E = 5?

E = 0.001, because fewer equivalent or better alignments are expected by chance.

Does the E-value directly measure percentage sequence identity?

No. It estimates the expected number of chance alignments with equivalent or better scores.

e value of 10

suggest you would expect to see 10 random hits of similar quality. not meaningful matches, unless query is very short

transcriptome

all of the expressed RNAs in a cell

does every cell have the same transcriptome?

NO! they vary based on several factors

what do transcriptomes vary by

cell type, tissue, organ, environemnt, treatment, time of day, and age

two technologies for transcriptomics?

microarray and RNA sequencing

microarray

1. obtain RNA by isolating

2. prepared labeled material

3. hybridization

RNA sequencing

1. polyA + mRNA extraction

2. fragmentation of mRNA

3. reverse transcription

4. size selection

5. sequencing of each end

What is the primary principle behind microarray technology?

Hybridization

What is the primary principle behind RNA-seq?

High-throughput sequencing.

Which technology provides single-base resolution?

RNA-seq

What is dynamic range in gene expression measurement?

The span of expression levels that a technology can quantify.

Which technology has a dynamic range exceeding 8,000-fold in the lecture comparison?

RNA seq

Which technology has greater ability to distinguish transcript isoforms?

RNA seq

What is a transcript isoform

A particular version of an RNA transcript produced from a gene, potentially through alternative splicing.

Which technology can distinguish allelic expression according to the lecture?

RNA seq

Which method requires less RNA input in the slide's comparison?

RNA seq

basics of rna sequencing

experimental procedure and contains rich information

Why is mRNA fragmented during the illustrated RNA-seq workflow?

To produce shorter fragments suitable for sequencing.

What is reverse transcription?

The conversion of RNA into complementary DNA (cDNA).

What is size selection?

Selecting prepared fragments within a desired size range.

What does paired-end sequencing produce?

Two reads originating from opposite ends of the same fragment.

ribo-minus

depletion strategy that removes abundant RNA populations

polyA

mRNA after splicing, enrich for exons, no r/tRNA, most popular

strand specific

directionality of RNA, useful for studying regulatory RNA

Strand-specific sequencing preserves

RNA directionality

depth

total number of reads

read length

Number of nucleotides in an individual sequencing read.