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How do you achieve cheaper, faster sequencing?

* The following article is written with excerpts from the second edition of The Sequencing Buyer’s Guide, written by Professor David Smith of the Mayo Clinic in Rochester, NY, and edited by Richard Lumb at Front Line Genomics. You can download the full version of the guide here. 

The field of genome sequencing is dynamic and constantly changing. It’s vital to keep up to speed, particularly as sample volumes increase and where even small improvements in cost, speed or efficiency can have a dramatic effect.

Here are just three important considerations for performing sequencing better, faster and cheaper.

1. Before you begin, it’s important to understand the strengths and weaknesses of the underlying technology.

For instance, short-read sequencing technologies like those provided by Illumina and BGI (as well as others) have the greatest sequence output at the lowest cost, as well as quite good sequencing accuracy, enhanced by both paired-end sequencing and higher coverage sequencing.

However, there are limitations. For instance, it can be difficult to generate complete sequence maps of complex genomes using short-read technologies, and it’s hard to determine which chromosome short reads are derived from.

Another problem with short-read sequencing is determining complex genome rearrangements, such as those that occur during cancer development.

Second generation sequencing technologies (long-read sequencing), from providers like Pacific Biosciences and Oxford Nanopore Technologies, solve many of the problems associated with short-read. Long reads make it considerably easier to assemble complete genomes. Complex rearrangements, including those frequently found in cancers, are much more easily characterised with this technology.

However, long-read technologies typically require high quality, long DNA fragments, which is not always possible, and the output on these machines is considerably lower than with short-read technologies that are available (although that is changing quickly).

You can read about others strengths and limitations of short- and long-read technologies by downloading the full guide here. 

2. Whole Genome Sequencing is the most expensive of many options. Ensure you consider other types of sequencing. 

Whole Genome Sequencing (WGS) feels like the obvious starting point as the genomics revolution progresses, but it’s not always the best answer.

While WGS provides the most complex information, it may be much more manageable and appropriate for your use-cases to use Whole Exome Sequencing (WES).

Likewise, Targeted Genome Sequencing can be much more cost-effective and quicker, such as the Foundation Medicine test developed to screen for mutations in over 300 cancer-related genes.

RNA Sequencing (RNA Seq), Methylation Sequencing and Microbiome Sequencing each also have their important uses.

To read more about which sequencing type is best for different use cases, download the guide here.

3. There are some easy ways to reduce the cost of your sequencing that you can adopt quickly.

The advances in increasing sequence output at a decreased price have been nothing short of phenomenal.

You can now generate 6-7 Tbs of sequence for the same amount of money that would only generated 20Mbs in 2006 – remarkable. And the genome of James Watson that was sequenced on the 454 platform in 2008 for 1 million dollars can today be performed on the Illumina NovaSeq for just $375!

But there are also some ways of reducing cost that are more within your immediate control – for example, in library preparation.

The most expensive MPS (Massively Parallel Sequencing) libraries to produce are those involved with WES or targeted genome sequencing, as the cost for the oligonucleotides themselves and the capture can be several hundred dollars. The generation of libraries for WGS is considerably cheaper as all you need to do is fractionate the DNA, repair the ends and litigate sequencing primers onto those fragments.

One approach towards decreasing the cost of sequence capture is to dramatically decrease the volume in which sequence capture occurs. There are several approaches towards this, but the most popular is based upon tiny water droplets in an oil emulsion.

In the same way that this works for emulsion PCR, you can put all the necessary oligonucleotide baits and total genomic DNA into droplets that have a volume in the nanolitre, or sub-nanolitre size range. This is the approach that was taken by BD/Gen Cell Biosystems. Since the volume is so much smaller this has two major advantages.

  1. It requires proportionally much less of the oligonucleotide baits.
  2. It also deceases the amount of genomic DNA needed for successful sequence capture.

The emulsion approach can also be used for other library preparations that are not dependent upon sequence capture and that can also both reduce the cost for library preparation and decrease the amount of starting DNA needed for library preparations.

To read more about other ways to dramatically reduce the cost of sequencing, download the full guide.

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