Whole genome sequencing is a powerful technology that is rapidly gaining popularity as it becomes
more and more affordable. With whole genome sequencing, you can assemble genomes de novo,
compare the genome of your organism to a reference genome, explore the molecular evolution of
a species or population, accurately track pathogen outbreaks, obtain the whole genome
sequence of an individual cell, and so much more. Whole genome sequencing begins with
the extraction of genomic DNA. This is typically done with a kit relevant to a specific sample
type, for example blood or plant tissue. The genomic DNA is made into libraries which are
quality checked, pooled, and normalized. They are then sequenced using an Illumina NGS platform
such as Nextseq. The amount of coverage and the number of reads needed will depend on
the type of genome and the application chosen. For a more detailed description of library
prep, quality control and coverage please see our previous videos.
Now let’s talk about some real examples of whole genome sequencing applications. Whole
genome sequencing can be used to assemble genomes de novo. Recently an international
collaboration achieved this for the genome of the flax plant and published in the plant journal
in 2012. Flax is an important crop both for food and textile production. The flax
genome was assembled using a short Shotgun Sequencing approach. The author’s generated
7 libraries with lengths varying from 300bp up to 10 kb pairs and sequenced them using
paired-end illumina. This generated mate-pair and paired end reads with 44 – 100 bp of
known sequence and a spacer of defined length. The use of mate-pairs from long libraries
replaces the conventional approach for correlating sequences long range. In the conventional
method, fosmid libraries are prepared in parallel to the NGS libraries and are sequenced using
Sanger Sequencing. This is more expensive and time consuming than the new NGS only approach
shown here. Aligning these reads generated contigs. The
contigs were further aligned to generate scaffolds. The scaffolds covered 85% of the predicted genome
length with 45X average coverage. For more details on how the genome was assembled, please
refer to the original publication. Expressed Sequence Tags or EST’s are short
sequencings obtained from cDNA libraries. They represent expressed regions of the genome
and so can be used to find protein coding genes. In this study, the known EST’s of
flax were aligned to the scaffolds. 93% of them aligned to the scaffolds with greater than
95% sequence identity, indicating that the assembled genes were highly accurate. For
more information about this study, please refer to the original publication.
Whole genome sequencing can also be used to track pathogen outbreaks. This proof of concept
study by Revez et al. does just that. Campylobacter jejuni is among the leading causes of food
borne illness in the world. It is naturally found in the guts of birds and cows. Humans
are most likely to become infected by ingesting contaminated water. C. Jujuni infection is
debilitating, but rarely fatal. In this study, samples from a C. jujuni outbreak in Europe
in 2012 were reanalyzed by whole genome sequencing and compared to the conclusions drawn from
standard methodologies. This was done in order to decide if whole genome sequencing had similar
or enhanced ability to track pathogen source and evolution during an outbreak situation.
The gold standard for analyzing strains of pathogenic bacteria is called pulsed-field
gel electrophoresis (PFGE), which compares the banding pattern between genomes digested
by a restriction enzyme. This approach is limited however, since significant mutations
can easily be hidden when they don’t affect the restriction sites or relative size of
the genomic fragments. At the same time, single nucleotide mutations can result in the gain
or loss of a restriction site and so can give a different PFGE pattern between closely related
strains. Based on the PFGE patterns observed during
the outbreak it was concluded that there was a contamination event involving one strain
and one water source. However whole genome sequencing revealed that this was not the
case. Of the two human isolates shown here, one was found to be highly similar to the
waterborne strain. The other, is highly divergent, too much so to be the result of genetic drift
during the course of the outbreak. In light of the whole genome sequencing data
the authors conclude that either a single source of water was contaminated by a multiple
divergent strains or that there were multiple sources of contamination. These results highlight
the importance of more accurate whole genome sequencing data during pathogen outbreaks,
since conventional methodology misidentified a patient strain, potentially missing an important
source of contamination. Whole genome sequencing is also an essential
tool for studying molecular evolution. This study, in Nature Communications in 2015, uses
whole genome sequencing to study the molecular evolution of the Ithica New York honeybee
population in response to the introduction of the mite Varroa destructor. Honeybees,
Apis mellifera, are essential to human agriculture. Both feral and domestic populations exist
in North America. Honey bees are a eusocial species. Each colony contains a sexually mature
queen bee, a few thousand haploid males and tens of thousands of sterile female worker
bees. The mite Varroa destructor feeds on the hemolymph of the adult worker bees, weakening
them and making them more susceptible to disease. The authors found a drastic loss of mitochondrial
haplotypes, with an entire clade going extinct. This loss indicates a population bottleneck
upon the introduction of Varroa destructor. However, they also found no decrease in nuclear
genetic diversity. This finding indicates that the modern population is descended from
a small number of queens through high rates of outbreeding and polyandry.
The ancestry of the modern bee population is similar to the museum bees with a few variations.
In the modern bees there are traces of African and Arabian ancestry that are absent in the
museum bees. The authors also found some genes that were under selection pressure in the
modern population relative to the museum bees. These genes may play a role in resistance
to the Varroa destructor parasite. For more details please see the original paper. This
study demonstrates how whole genome sequencing is a powerful tool for studying the molecular
evolution of a population over time. This has been just a few applications of
whole genome sequencing. Thank you for watching. Look forward to our next video coming soon,
and don’t forget to leave your questions and comments below.