6. NGS: read mapping

Question 1

What is the similarity/difference between assembly and read mapping?

Answer 1

Assembly: first genome of a species
- de novo assembly (high quality assembly: expensive/difficult)
- genome structure, gene inventory, etc

Read mapping: next genome(s) of that species (or a close relative)
- de novo assembly serves as reference: read mapping, re-sequencing, alignment
- cheaper: lower coverage required
- different questions

Question 2

What is read mapping aka

Answer 2

AKA
mapping, alignment, read sequencing

Question 3

What is reference bias?

What is a possible solution?

Answer 3

Mapping bias / reference bias

• reads containing non-reference alleles
  
  • might not be mapped or incorrectly mapped
  • receive lower mapping scores (mismatches!)
• effect increases with genetic distance from the reference genome
• affects downstream analysis (e.g., population genomics)

solution
* map to multiple reference genomes

Question 4

NGS read mapping - approaches?

Answer 4

1: (pre-proccess &) identify short matches
* hash-table based (Sanger, NGS)
* BWT based (NGS)

2: extend seeds to longer alignments (e.g., DP)

Question 5

What is a pan-genome?

Answer 5

collection of common and unique genomes present in given species.

combines genetic information of all genomes sampled –> large and diverse range of genetic material

Question 6

What is a suffix tree?

Answer 6

compressed trie containing all suffixes of the given text as their keys and positions in the text as their values.

Question 7

define genotype

Answer 7

In a broad sense, the term “genotype” refers to the genetic makeup of an organism; in other words, it describes an organism’s complete set of genes. In a more narrow sense, the term can be used to refer to the alleles, or variant forms of a gene, that are carried by an organism.

Question 8

What is a hash table

Answer 8

eg dictionary in python

Question 9

How is hashing used for read mapping?

Answer 9

• create hash table with substrings of length k called k-mers extracted from reference genome as keys, and their positions on the reference as values
• Then, some k-mers from each read are looked up in the hash table to find candidate locations of the read on the reference (hits)

Question 10

In hash -table based read mapping, what are the drawbacks of short kmers? of long kmers?

What is the solution?

Answer 10

smaller templates (substrings, k-mers)
- allow to map in the presence of more errors/variation
- are not very effective seeds (match too many regions)
- dilemma!

solution: spaced seeds

Question 11

What are spaced seeds?

Answer 11

templates: optimized for a given read length and
expected error/mismatch rate

pattern of relevant and irrelevant positions in a biosequence and a method of approximate string matching that allows for substitutions.

usually represented as a sequence of zeroes and ones

Question 12

consecutive seeds vs. spaced seeds

Answer 12

consecutive seeds: one template, needs contiguous matches

spaces seed: template allows mismatches

Question 13

What is a BW matrix?

Answer 13

BW matrix:
rows are sorted cyclical rotations of string$ - equivalent to sorting the suffixes

Question 14

What is BWT?

Answer 14

Burrows-Wheeler Transform
- full-text indexing approach
- suffix array-like index structure

Question 15

What are the advantages of BWT-based approaches for read-mapping?

Answer 15

• space efficient, can be efficiently compressed
• original string can be recovered
• can fit in RAM of most computers
• pre-computed indices can be stored & shared

Question 16

What are the most widely used read mapper tools?

Answer 16

BWA, Bowtie2

Question 17

What problem does mapping of long reads present and how can this be addressed?

Answer 17

• higher number of sequencing errors (and different types of errors)
• hash-based and BWT-based approaches
• longer reads: many short seeds per read
• higher error rates –> use chaining
• use representative seeds only (minimizers)

Question 18

What is SAM and BAM?

Answer 18

Alignment output
* compact file size
* analysis without loading the alignment into memory
* can be used by alignment viewers (IGV, samtools)
* SAM (Sequence Alignment/Map) format - human readable (sort of), can be parsed easily
* BAM format - same information but in binary format

Question 19

In SAM format, what is the flag code?

Answer 19

tell us if the strand is mapped, (un)paired, etc

Question 20

What is mapping quality?

When is this relevant?

Origin? Usage?

Answer 20

mapping quality= probability of read aligned to incorrect region

Relevant with repetitive genome regions, SNPs, sequencing errors
➡ reads map equally well to multiple positions
➡ reduced probability of assigning a read to the correct genomic location
➡ measure the confidence that a read actually comes from the position it is aligned to

first introduced with MAQ mapping software (2008)
different programs compute this differently! (and some don’t compute it at all)

Question 21

What is a single bp variant? What is an SNP?

Answer 21

SNPs
* a single bp change with a frequency ofat least 1% within a population
* bi-allelic

others: rare, just called s bp variant

Question 22

What can cause single bp variants?

What do we need to consder in order to resolve this?

What approaches?

Answer 22

Cause:
errors, paralogs, diploidy, polyploidy, …

considerations:
read depth, read quality, alignment quality, frequency and ratio of SNPs, number of equally good alignments per read, …

approaches
- allele counting
- probabilistic methods

Question 23

What is variant calling?

Name one package and the tools to do this

Answer 23

Variant calling entails identifying single nucleotide polymorphisms (SNPs) and small insertions and deletion (indels) from next generation sequencing data

bcftools

1. bcftools mpileup
   * for every position, considers data from all reads covering the position
   * calculates genotype likelihoods
   - based on mapping qualities of the reads, base qualities, the probability of local misalignment, per-base alignment quality (BAQ)
2. bcftools call
   * calls the most likely genotype under the assumption of Hardy-Weinberg equilibrium, using allele frequencies estimated from the data
   or provided explicitly by the user

Question 24

What is VCF?

Answer 24

Variant Call Format
* text based
* meta-information, genome variation by position
* can include info about structural variation

Question 25

What are structural variants?

size?

relevant for?

etc

Answer 25

variation generally >50bp

~2% of the human genome

relevant for medicine, molecular biology, evolution

least studied type of variation, because they are very difficult to detect with short reads

Question 26

What are different types of structural variants?

Answer 26

• insertions, deletions, duplications
• inversions, rearrangements
• copy number variations

Question 27

Structural variants with:

short reads
long reads
genome alignment

Answer 27

short reads
- need to take into consideration distance and orientation of
paired reads; split reads
- some specialized mappers exist
- none of them can identify all SV types
- limit inherent in the short reads

long reads
- in theory advantageous, span SV regions
- in practice: not very many methods yet available
- often specific for sequencing technology

genome alignment
- required: de novo assembly, spanning the SV
➡ useful & important! methods are still evolving!

Question 28

Describe fasta format

Answer 28

• text-based format
• representing either Nts or AA using single-letter codes.
• begins with a single-line description
• followed by lines of sequence data

Question 29

Describe fastq format

Answer 29

• text-based format
• for storing biological sequence + corresponding quality scores.
• Both encoded with a single ASCII character

Question 30

EXAM QUESTION

Describe 2 structural variants and how to detect them during mapping of long-read sequencing data (2022)

Answer 30

• insertions
• deletions
• duplications
• inversions, rearrangements
• copy number variations

long reads are split into sub-reads and mapped - anlaysis can then determine if they are one of the SVs above