De Novo Assembly

Question 1

What are mate-pairs (MP)? What types there are?

Answer 1

• sequencing the two ends of genomic fragments
• usually considered only reads at unique positions
• long sequences
• two fragments that are distal to each other in the genome and in the opposite in orientation to that of a mate-paired fragment
• Paired-out
  
  • mates map on 2 different contigs -> merge and assess gap
• Paired-in
  
  • mates map on the same contig -> validate assembly, identify structural variations
• Single
  
  • one read maps and its mate-pair does not -> close to a gap

Question 2

What is a contig?

Answer 2

• series of overlapping DNA sequences

- merged makes a contigous fragment of DNA

Question 3

What are Structural variations (SV)?

Answer 3

• genomic differences involving large segments of DNA
  
  • deletions
  • insertions
  • inversions
• can be normally occurring in the population or pathological

Question 4

What is scaffolding?

Answer 4

• linking together a non-contiguous series of genomic sequences into a scaffold
  
  • bridge gaps between contigs

Question 5

What can Pair-end and Mate-pair sequencing be used for?

Answer 5

• scaffolding (and gap filling)
• validation of de novo genomic sequencing
• structural variations detection
• two things needed: a genome and the mate paired reads

Question 6

What is physical coverage?

Answer 6

• average number of times a base is read or spanned by mate paired reads
• mate pairs obtained from long physical inserts would be most effective for scaffolding

Question 7

What is sequence coverage?

Answer 7

• average number of times a base is read

Question 8

What does pair-end mean?

Answer 8

• short sequence

* set of fragments read on both ends

Question 9

Detection of structural variations with mate pair libraries

Answer 9

• insert length statistics for the identification of structural variations
• Use of “broken” reads to identify points of
  insertion/deletion
  
  • should result broken wher junction occurs

Question 10

Sanger sequencing

Answer 10

• 1977, Frederick Sanger
  
  • fluorescent dyes, first “automatic” DNA sequencers appeared
  • quality deteriorates with length of fragment
• based on possibility of creating a lot of subfragments of the region to be sequenced and many copies
  
  • start at same position
  • termination should be according to 4 classes, random base, dideoxy terminator

Question 11

Varible loci, what are those? What is the difference between polymorphism and mutation?

Answer 11

• in some individuals a locus could be found in 60% of the individuals as a C and in 40% as a T
• about 1 out of 500 basis
• polymorphism -> at least in 1% of population
• mutation -> less than 1%

Question 12

What are the problems and solutions of whole genome sequencing?

Answer 12

• genomes of million/billions bases long
• Sanger -> 500/1000 bases long reads
• possible solutions:
  
  • generate contigours fragments and sequence them (unfeasible)
  • shotgun approach (sequence random fragments)

Question 13

Hierarchical shotgun assembly

Answer 13

• BACs (Bacterial Artificial Chromosomes) can host 150 kbp of DNA
• transfered into E. coli they replicate
• BAC clones are sequenced independently, either randomly or to obtain minimum overlap
• assemblying in difficult (NP hard) Hamiltonian path
• Gaps:
  
  • some regions could not be covered
  • repeats make association of contigs difficult

Question 14

Poisson distribution

Answer 14

• f(v) = (e^-r * r^v)/v!
  
  • f(v) expected frequency that a base is found v times
  • r rendundancy, average coverage
• 1-e^-r, part of the genome covered at least one time

Question 15

Better assembly strategies

Answer 15

• from shotgun reads to contigs
• Greedy algorithm
  
  • no need to calculate all possible paths
• 1/2 *n^2 possible overlaps calculated, n possible reads
• Process:
  
  • pairwise alignments of all fragments
  • choose two fragments with the largest overlap
  • merge
  • repeat until only one fragment left
• complexity increases as n^2

Question 16

Scaffolding

Answer 16

• from contigs to scaffolds
• mate-pairs:
  
  • pairing the ends can help merge contigs and resolve difficult repeated regions
• impossible to complete a complex genomic sequence with the only approach of sequence overlaps

Question 17

What are the uses of mate pairs?

Answer 17

• Scaffolding
  
  • unique pair out quite useful in closing gaps
• Assembly validation [contigs]
  
  • mates align on both contig and distance compatible with distance of genomic insert
• Gap closure

Question 18

Read assembly and Eulerian paths

Answer 18

• Graph, reads vertices and read overlaps arches, occurrences
  
  • sequences occurring at a higher rate may be repeated more than once
• Eulerian path, similar but easier to compute
  
  • De bruijn graphs, kmer of length k, one-out-one-in
  • long enough, should’t be repeats (present only once)

Question 19

De Bruijn graphs practical application

Answer 19

• shotgun library sequenced at high coverage (60x)
• fastq -> kmers are counted (kmers and frequency)
• start from any kmer, extend one position at a time looking at list:
  
  • no repeats -> only one present
  • few counts could be errors
  • more counts could be repeats (more edges)

Question 20

De Bruijn graphs and mate pairs

Answer 20

• same fragment could be sequenced from both ends
  
  • inverted sequences should be counted as one
• kmer best length is l20/30 bases
• genomics sequences -> lots of repeats -> many branches
• using mate pair libraries could help identify the right path