Methods for sequencing of nucleic acids

Question 1

What does DNA stands for, and which four bases makes up the DNA?

Answer 1

DNA = Deoxyribonucleic acid.
Four nucleotides are the basic units of DNA molecules:
1) Adenine
2) Guanine
3) Cytosine
4) Thymine

Question 2

What is DNA sequencing?

Answer 2

DNA sequencing is to precisely determine the order of the nucleotides adenine, guanine, cytosine, and thymine in a DNA molecule.

Question 3

Which factors is included in a Sanger Sequencing?

Answer 3

1) Amplified template (unknown) DNA
2) DNA polymerase
3) Primers
4) Nucleotides: dATP, dTTP, dCTP, dGTP
5) Dideoxy/chain-terminating versions of all nucleotides: ddATP, ddTTP, ddCTP, ddGTP

Question 4

What is the method of Sanger Sequencing?

Answer 4

When all ingredients are added to the mixture, the polymerase starts to grow new DNA strands which are complementary to the original DNA template.
This process is ongoing until a dideoxynucleotide incorporates - randomly.
When this happens, no further nucleotides can be added, and the strand stops to grow.
When the sequencing reaction is finished, DNA fragments of different lengths have been synthesized. A dideoxynucleotide will be represented in every position of the original DNA.

Question 5

How is capillary gel electrophoresis performed?

Answer 5

The fragments from the Sanger Sequencing are sorted according to size in a process called capillary gel electrophoresis. The smallest fragment crosses the finish line first, followed by the next-smallest fragment and so forth.
When each fragment crosses the finish line, its illuminated by a laser, allowing the attached dye to be detected.
The data recorded by the detector consist of a series of peaks in fluorescence intensity. The DNA sequence is read from the peaks in the chromatogram.

Question 6

What is advantages and limitations of Sanger Sequencing?

Answer 6

Advantages is the high quality, and that is can sequence long sequences (~900bp).
Limitations are high cost, and low capasity.

Question 7

Which methods is included in second generations platforms?

Answer 7

Platforms are Ion Torrent, and illumina sequencing.

Question 8

What is Shotgun Sequencing?

Answer 8

A sequencing method where a large DNA molecule is fragmented to short sequences. These fragments are further assembled by a data program of overlapping DNA sequences.

Question 9

How does amplification of sequencing libraries occur?

Answer 9

1) ssDNA anneals to primers on a flow cell
2) Polymerase starts amplification from the primer
3) Removal of original single strands, the amplified strand is physically attached to the flow cell
4) The other end of the DNA anneals to the flow cell. A “single stranded bridge” is formed
5) Polymerase starts amplification from the annealed side. A “double stranded bridge” is formed
6) Denaturation of the “double stranded bridge”. The two complementary strands are attached to the flow cell (a cell each)
7) At the end of the amplification, millions of clusters are formed on the flow cell
After amplification and denaturation: both forward and reverse DNA in cluster. Reverse strand is removed. Only forward strand is sequenced.

Question 10

Which length can be sequenced with Illumina, Ion Torrent, and Sanger?

Answer 10

300 bp Illumina
400 bp Ion Torrent
700-800 bp Sanger

Question 11

What is the process of Whole Genome Sequencing (WGS)?

Answer 11

1) DNA Extraction.
Scientists take bacterial cells from an agar plate and treat them with chemicals that break them open, releasing the DNA. The DNA is then purified.
2) DNA Shearing.
DNA is cut into short fragments og known length, either by using enzymes “molecular scissors” or mechanical disruption.
3) DNA Library Preparation.
Scientists make many copies of each DNA fragment using PCR. The pool of fragments generated in a PCR machine is called a “DNA library”.
4) DNA Library Screening.
The DNA library is loaded onto a sequencer. The combination of nucleotides making up each individual fragment of DNA is determined, and each result is called a “DNA read”.
5) DNA Sequence Analysis.
The sequencer produces millions of DNA reads and specialized computer programs are used to put them together in the correct order like pieces of a jigsaw puzzle. When completed, the genome sequence containing millions of nucleotides is ready for further analysis.

Question 12

How is the preparation of a Nanopore library performed?

Answer 12

High molecular weight gDNA
1) Optional fragmentation
2) Combined FFPE repair and end-prep
3) Ligation of barcodes
4) Ligation of sequencing adapters

Question 13

What is Nanopore Sequencing?

Answer 13

At the heart of the MinION device, an enzyme unwinds DNA, feeding one strand through a protein pore. The unique shape of each DNA base causes a characteristic disruption in electrical current, providing a readout of the underlying sequence.

Question 14

How is the Pacific Biosciences library prepared?

Answer 14

1) Fragment DNA and determine concentration
2) DNA Damage repair
3) Repair ends
4) Ligate adapters
5) Purify templates
6) Primer annealing
7) Bind polymerase and sequence

Question 15

What is de novo assembly?

Answer 15

The process of reconstructing the original DNA sequence from the fragments alone.
1) Fragment DNA and sequence
2) Find overlaps between reads
3) Assemble overlaps into contigs
4) Assemble contigs into scaffolds
The process is easier if you have longer and fewer reads, compared to smaller and more reads.

Question 16

What is metagenomics?

Answer 16

Metagenomics is the sequencing and analysis of genomic information extracted directly from clinical or environmental samples, without the need of culturing

Question 17

Which three revolutions has genome sequencing been through?

Answer 17

1) Whole-genome shotgun sequencing
2) High-throughput sequencing
3) Single-molecule long-read sequencing

Question 18

What is the genome characteristics of E.coli and Enterobacteriaceae?

Answer 18

E.coli is one of the most studied organisms in the world. Its genome size ranges from 4.6 Mb to 5.9 Mb for a median GC content of 50.6%, with 4200 to 5500 genes. Overall, Enterobacteriaceae are characterized by a large variable genome with various intrafamily horizontal gene transfer (HGT) or recombination, sometimes increased by the host`s medical conditions.

Question 19

What does MLST stands for, and what is it?

Answer 19

MLST stands for “Multi-locus sequence typing”.
It is an unambiguous procedure for characterising isolates of bacterial species using the sequences of internal fragments of (usually) seven house-keeping genes. Approximately 450-500 bp internal fragments of each gene are used, as these can be accurately sequenced on both strands using an automated DNA sequencer. For each house-keeping gene, the different sequences present within a bacterial species are assigned as distinct alleles and, for each isolate, the alleles at each of the seven loci define the allelic profile or sequence type (ST).

Question 20

What is the genome characteristics of Staphylococcus aureus and coagulase-negative staphylococci?

Answer 20

Staphylococcus aureus has a genome size that ranges from 2.6 Mb to 3.1 Mb, with a median GC content of 32.8%. Coagulase-negative staphylococci (CoNS) have similar genome features to S.aureus. Mobile genetic elements represent 15-20% of the S.aureus genome, emphasising the important transfer of virulence factors and/or antimicrobial resistances that can happen between strains or even between species.

Question 21

What is SCCmec?

Answer 21

A mobile genetic element carrying the methicillin resistance gene in S.aureus.

Question 22

What is the genome characteristics of Streptococci and enterococci?

Answer 22

The median lengths are 1.8 Mb and 2.1 Mb for Streptococcus pyogenes and S.pneumoniae, respectively. Enterococci of medical importance have larger genomes, ranging from 2.6 to 3.4 Mb. The GC content for these varies from 35% to 40%. Overall, streptococci and enterococci display high genome plasticity. HGT and homologous recombination can drive serotype modifications, as well as the spread of virulence factors and antibiotic resistance genes.

Question 23

What is the genome characteristics of Pseudomonas aeruginosa and Acinetobacter baumannii?

Answer 23

The P.aeruginosa genome size ranges from 6.1 to 7.5 Mb, with a median GC content of 66.2%. For A.baumannii, its genome size is shorter and varies from 3.7 to 4.3 Mb, with a median GC content of 39%. HGT and genome-wide homologous recombination plays a major role in these two successful and often multidrug-resistant opportunistic pathogens. Plasmid-mediated antibiotic resistances play a major role in the transmission of antimicrobial resistances between isolates and species, which may be hard to assess based only on short reads sequencing.

Question 24

What is the genome characteristics of Mycobacterium tuberculosis?

Answer 24

Mycobacterium tuberculosis complex (MTBC) has a clonal, monomorphic genome of approximately 4.3 to 4.4 Mb. HGT or recombination do not occur in MTBC, whereas it is an important driving force for evolution in other mycobacteria. Thus, antimicrobial resistances can only occur from SNPs or insertion-deletion events in MTBC.

Question 25

What is the genome characteristics of Chlamydia trachomatis?

Answer 25

Chlamydia trachomatis has a small genome size, as a consequence of the adaptation to its intracellular habitat, of 1.0 Mb to 1.1 Mb, with a median GC content of 41.2%. Although there are evidences for HGT and especially for homologous recombination, these mechanisms seem to play a smaller role that point mutations for driving the evolution of C.trachomatis.