Genomic
Sequencing is an exciting field of development. Starting with
the first generation of sequencing, Sanger sequencing in the 1970’s, the last decade and a half has
seen the release of two more generations.
Second generation or Next Generation Sequencing (NGS) took off in the late 2000’s/early 2010’s and presented a
methodology to sequence an entire genome using short-read technology. It is the
main sequencing methodology used today and Illumina are the major providers,
accounting for the majority of the world’s sequencing data.
A few years later the third generation or long-read
sequencing methodologies were launched, producing technologies that sequence
significantly longer DNA fragments and hence can cover pesky repetitive DNA
regions and offer the ability to perfectly sequence a genome.
While these technologies were first developed over a decade ago, the current state of affairs in 2nd generation (NGS) and 3rd generation (long-read) sequencing is by no means at the peak. For one, we’ve seen a gradual improvement in sequencing accuracy over recent years, and it is at its highest point today.
The genome, or genetic material, of an organism is
made up of a unique DNA or RNA sequence. Each sequence is composed of chemical
building blocks known as nucleotide bases. Determining the order of bases is
called "genomic sequencing" or, simply, "sequencing."
The information encoded in the genomes of
disease-causing bacteria, viruses, and fungi represent unique genetic
fingerprints. Whole-genome sequencing (WGS) is a laboratory procedure
that determines the order of all, or most, of the nucleotides in the genome of
these disease-causing microbes. By examining the sequence data at a community
level, public health officials can better understand how microorganisms move
through populations and change over time.
Next-generation sequencing (NGS) refers to sequencing technologies that can process a large quantity of genetic material at a time. These technologies have been available since 2004 and have largely replaced the previous method (Sanger sequencing). NGS technology makes high-throughput WGS possible. Newer sequencing platforms have revolutionized this field by generating larger volumes of data and dramatically lowering the cost of sequencing.
The genome sequencing steps
Step 1: Extraction
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| DNA must be extracted from the cell. |
Strands of DNA or RNA are first extracted from the
bacteria, virus, or other pathogen.
Step 2: Library Prep
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| DNA is cut before sequencing. |
The DNA or RNA to be sequenced must be specially
prepared before it can be put into the sequencing machine. The steps in the
process may differ depending on the type of sample and specific equipment being
used. Some of the steps include:
Converting RNA or single-stranded DNA into
double-stranded DNA if needed
Chopping strands into shorter pieces to get a desired
length
Modifying the ends of the fragments so that they can
be recognized by the sequencer. At this point, the sample is called a
"library" and is ready for sequencing.
Step 3: Sequencing
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| Sequencers use |
The library is loaded into a sequencer, which
identifies the nucleotide bases in the DNA fragments. Some next-generation
sequencing devices read fluorescent signals. Others identify bases by
monitoring changes to an electrical current as the DNA strands pass through
tiny holes.
Step 4: Analysis
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The sequencer produces data—millions of long strings of letters—that are then assembled together or aligned with a reference sequence. Analytical programs compare the new sequence data to the reference sequence and identify variations in the sample that allow scientists to infer an ancestral relationship, much like a family tree.
New pathogens can be identified by comparing their sequence to a database of all known pathogens and finding related species.
