4 Approaches to HLA Type using Next Generation Sequencing

Human leucocyte antigen (HLA) genes are among the most polymorphic in the entire genome. They are located on the short arm of chromosome 6 within the major histocompatibility complex (MHC) and play an important role in organ or hematopoietic stem cell transplantation. Donor – recipient matching prior to transplantation is performed by examining 6 genes (A, B, C, DP, DQ, DR) that encode HLAs to reduce risk of transplant rejection or graft-versus-host-disease (GVHD).

 While the current “gold-standard” for HLA typing is Sanger Sequencing, defining the phase of sequence motifs is difficult and genotype ambiguity increases with each database release. Next generation sequencing (NGS) offers many advantages including: the ability to provide nucleotide-specific reads across the entire genome, clonal amplification provides phase information, and the ability to thoroughly evaluate larger genomic regions (introns). Unresolved technical issues however have limited the implementation of NGS in accredited HLA laboratories. These include significant library preparation and processing times and the more complex bioinformatics analysis needed to generate an easily readable HLA typing report.

 The main approaches to prepare DNA template for HLA typing on NGS platforms include:

  1. Multiplex PCR to amplify target regions, including exons or introns. Advantage: After PCR, fragmentation and ligation are no longer required as sequencing primers are designed as past of the initial targeting primers.  Disadvantage: Primer design is difficult due to multiple polymorphisms, necessitating primer pools.
  2. Long range PCR of individual loci followed by fragmentation and ligation of sequencing adapters. Advantage: Longer regions can be targeted. Disadvantage: Fragmentation and subsequent ligation of sequencing adapters can be difficult.
  3. Sequence capture using oligo based hybridization to target regions >20 Mb. Advantage: This technique is similar to exome capture and well characterized. Disadvantage: Hybrid capture of shorter HLA regions or the MHC is less effective than larger segments.
  4. Sequencing of the entire genome (whole genome sequencing). Advantage: This is the least biased way to examine HLA regions of interest. Disadvantage: Data analysis and the requirement to extract sequences of interest from the entire genome is difficult and not ready for routine studies.

Generating an unambiguous HLA genotype is important and software customized to each of these 4 approaches now exists. Several commercial companies, including bioinformatics providers on Genohub have software to accept data in FASTA or FASTQ and output accurate HLA genotype results.

 Whether you’re just beginning your HLA typing work and are interested in library prep, sequencing and an analysis solution or if you’ve already extracted your data and now are looking for the right analysis to generate an unambiguous report, Genohub offers complementary HLA consultation and can match you with the right service provider. Service providers on Genohub have experience using the four template preparation methods described above and have the pipelines in place for your analysis. To get started, fill out our NGS project consultation form and we’ll contact you with our recommendations. 


Bioinformatic Analysis: A New Option On the Genohub NGS Market

The ability to list and purchase bioinformatic analysis services along with next-gen sequencing comes to Genohub. This additional feature set will further simplify the NGS project experience by offering capabilities for the bioinformatics project step, all in one roof. By launching this service, researchers on Genohub are now able to shop from full-service sequencing providers who offer the three main services required in every NGS project with options for Illumina, Ion, SOLiD, PacBio, and Roche454 platforms:

  1. Library prep
  2. Next generation sequencing
  3. Bioinformatic analysis

The addition of this service alleviates the burden many researchers face of shopping for three separate sequencing related services from multiple providers and efficiently managing the entire process.

The benefits of this feature for researchers includes:

  • Researchers save time and money purchasing sequencing and bioinformatic services simultaneously
  • By enabling next gen sequencing providers to list bioinformatic services, it becomes easier and faster for researchers to compare and select a provider for their project
  • Risk is mitigated, as researchers are able to purchase bioinformatic services along with NGS services by providers that have gone through the Genohub vetting process

Providers also benefit:

  • Providers are now able to better highlight their unique services by listing specific bioinformatic services. These include basic primary analysis that may be included with every sequencing order, as well as more complex secondary and custom bioinformatics services that can be ordered as add-ons to NGS projects.
  • The bioinformatics platform enables providers to more succinctly communicate bioinformatic services to researchers, cutting down on back and forth communication prior to the researcher opting to move forward with the services listed by the provider

Providers may now list bioinformatic analysis services on the familiar “Manage Services Offerings” page:

New manage services page includes bioinformatics analysis services

New Manage Services Page

Researchers will be able to view these services once they select to view details about a sequencing package found when shopping by project or technology.

At Genohub we constantly strive to improve the service we offer to the NGS community through the development of new features and functionality. When we launched Genohub in August, 2013 our goal was to launch a service that was useful to researchers and sequencing providers, and continue to enhance and improve our service based directly on feedback from our clients.

The bioinformatic analysis listing feature come as a direct result of our development methodology. Researchers asked for a one-stop-shop for all of their sequencing needs, and sequencing providers asked for more ways to highlight and differentiate their services on Genohub. You asked, we listened!

Please visit Genohub often as we are constantly adding new useful features to improve the next-gen sequencing shopping experience including upcoming features to further enable providers to differentiate themselves and mitigate risk in decision making for researchers. If you have any feedback on additional features you would like to see, please send us feedback by shooting us an email at info@genohub.com.

In a Nutshell: Life Tech Exome Certified Service Provider Program

Life Technologies announced yesterday that they launched the Ion AmpliSeq Exome Certified Service Provider Program.

What the program is in a nutshell:

  • Goals: Offer a network of next gen sequencing providers able to help researchers get a high quality exome sequence at a reduced cost with fast turnaround times and low amounts of input material
  • Exome sequencing inputs: as little as 50ng of customer DNA
  • Library kit used: Ion AmpliSeq Exome kit
  • NGS Instrument used: Ion Proton
  • Exome sequencing outputs: high quality data, which of course can be used with Ion Reporter Software for mutation validation, annotation, and reporting

The Service Provider Program is intended to fill exome sequencing market demand which Life Tech argues has been under-serviced with exome sequencing currently going for $1,000+ , long turnaround times up to 8 weeks, and requiring up to 3mg of DNA. Dr. Candace Johnson, Deputy Director and the Wallace Chair of Translational Research at Roswell Park Cancer Institute states “Exome sequencing will be central to discoveries made in clinical research”. If the Exome CSP delivers as promised, it could have a major impact in accelerating discoveries made in clinical research.

For more information on the Life Tech Provider Program please see the entire press release.

Targeted Resequencing (TPS/WES) Tops Next Gen Sequencing Survey

Oxford Gene Technology (NGS provider currently listed on Genohub) recently presented the results of their next gen sequencing survey which demonstrated targeted resequencing as the top use for next generation sequencing. The results are based on a survey of 596 researchers who responded regarding their current and expected use of NGS services. When compared to the results for whole genome sequencing the popularity of targeted resequencing is possibly attributed mostly to the lower cost of targeted resequencing. This infographic depicts the results:

OGT NGS Survey Results

OGT NGS Survey Results

Other interesting results point to a general data problem with 38% of respondents saying they lack trust in bioinformatics data. Bioinformatics also leads the field when researchers were asked about the biggest barrier to NGS usage (see below).

Barriers to NGS Usage

Barriers to NGS Usage

Undoubtedly this presents an immense opportunity for the bioinformatics sector to increase confidence in data accuracy and interpretation which could have a positive impact on the use of next gen sequencing as a whole.

You can find many more interesting survey results on the excellent infographic titled Oxford Gene Technology – NGS Survey 2013.

First XPRIZE Cancelled Due To Unexpected Innovation in Next Gen Sequencing

For the first time ever, an XPRIZE has been cancelled. The reason — unexpected innovation in next generation sequencing. The Archon Genomics XPRIZE announced in 2006, had promised to award $10 mil to the first team that was able to accurately sequence 100 whole human genomes at a cost of $10,000 or less per genome in a short period of time. The competition was cancelled as XPRIZE CEO Peter Diamandis and team felt it was not serving its intended purpose to incentivize technological innovation in gene sequencing.

As stated by Peter Diamandis, “Every XPRIZE is carefully designed to address a market failure and hopefully create a new industry to achieve breakthroughs and solutions once thought to be impossible.” Although the Archon Genomics XPRIZE was conceived according to this criteria, the XPRIZE team felt that innovation in gene sequencing has been progressing independently of the XPRIZE incentive, therefore voiding the need for the competition.

The rapid innovation in next generation sequencing has caused sequencing times to decrease and prices to plummet to around $5,000 per genome. The XPRIZE team feels as if the targets laid out by the competition will be met in the very near future with or without their incentive, and have opted to cancel the XPRIZE and return the money to sponsors. The announcement by Peter Diamandis can be read in its entirety on the Huffington Post.

The logic behind the XPRIZE cancellation seems clear, however it remains to be seen what backlash, if any, arises from scientists who may have spent considerable time and effort devoted to meeting this challenge. Although next gen sequencing instruments are developed by large companies such as Illumina ($1.15B revenue), which may not be driven by a competition like the XPRIZE, innovation in this field must also be attributed to the wider research community, of which a team may have conceivably won the competition independent of any large commercial enterprise. In fact, in his cancellation announcement, Peter Diamandis thanks George Church and the Wyss Institute at Harvard for registering for the competition. Does the XPRIZE lose some of its ability to incentivize future competitions because of this cancellation? We welcome your comments on the matter.

2 Ted Talks on the Next Generation Sequencing Impact

Here are a couple of great Ted Talks on the impact of next generation sequencing on a variety of areas that promise to fundamentally change our daily lives.

This is a good talk about the future of sequencing and the impact of massively parallel sequencing. The impact to patient care, food supply, health insurance, politics, and more are discussed.

Richard Resnick: Welcome to the genomic revolution:



More of an introductory discussion on gene sequencing. What is genomics? How will it affect our lives? In this intriguing primer on the genomics revolution, entrepreneur Barry Schuler says we can at least expect healthier, tastier food. He suggests we start with the pinot noir grape, to build better wines.

Barry Schuler: Genomics 101:



Next Generation Sequencing Creates Opportunities for Software Entrepeneurs

Earlier this year it was announced that a large biotechnology firm, Qiagen based in the Netherlands completed its purchase of Ingenuity Systems based in California which had built software exclusively. This event is one of many to signal the trend of a merging of the biotechnology and software industries which is presenting a large opportunity for software entrepreneurs.

This trend, as further discussed on the Washington Post, is being led by the next generation sequencing industry which continues to produce sequencing data at ever increasing rates and at lower costs. As sequencing data becomes more readily available, the demand continues to rise as some experts suggest DNA mapping will become standard medical practice. Previously it had been difficult for software companies to thrive in the biotech space as next generation sequencing hadn’t come along until massively parallel signature sequencing (MPSS) was developed in the 1990’s. Because previous methods were costly and not readily available, the demand for sequencing analysis was held equally low.

As vast amounts of next generation sequencing data are now produced at ever quicker rates, there arises a tremendous opportunity for software entrepreneurs who are able to produce usable results from this data. Although the industry is in its infancy, and has not yet produced a clear market winner, there are several new companies which are capitalizing on this demand. One of these is Enlis Genomics which was started two years ago in Berkeley, CA by Devon Jensen, a PhD in molecular and cell biology. Devon saw the demand for software which was easy enough to use by the average researcher. Another notable example would be Cypher Genomics out of San Diego, CA also formed two years ago which allows researchers to detect genetic variations which may signal health concerns.

Undoubtedly we will also continue to see biotech firms of all types continue to gobble up these new bioinformatics companies as they strive to offer a complete front to back solution for their clients. This is truly an exciting time in the bioinformatics industry with a host of opportunities yet to be discovered.

Scientists Develop Method to Rapidly Analyze High Throughput Sequencing Data

Scientists in Singapore have developed a method to rapidly analyze high throughput sequencing data. This method incorporates a mathematical technique which has long been used in industries outside biotechnology such as radar, electrical engineering, and cell phones.

While high throughput sequencing has revolutionized molecular biology, there has been a speed bump which slows the analysis of sequencing results. The high throughput sequencing process generates a massive amount of data which must be analyzed to filter signal from noise. Traditionally, researchers have treated each set of sequencing data as unique and requiring its own set of analytical methods to decipher. Because of this, it can take a lengthy period of time to produce meaningful sequencing data.

As published in Nature Biotechnology, the team of scientists was able to produce meaningful results from a variety of high throughput sequencing data sets using only one analytics technique, the pre-whitening match filter. The filter was demonstrated to produce data across several sequencing based functional profiles that is more accurate than assay-specific analysis methods.

This latest technique holds promise to reduce time and cost for patients who could benefit from an improved level of care due to sequencing.

The article can be found at: Kumar et al. (2013) Uniform, Optimal Signal Processing Of Mapped Deep-Sequencing Data.


Next Generation Sequencing Promises to Increase IVF Success Rates

Next generation sequencing techniques hold promise for increasing the success rate of in-vitro fertilization (IVF). Dr Dagan Wells of the NIHR Biomedical Research Center at the University of Oxford announced on July 8, 2013 at the ESHRE annual meeting that an IVF birth had resulted, for the first time, due to embryo selection based on next generation sequencing results.

Historically, IVF success rates have stood at around 30%, thought to largely be due to DNA mutations in the embryo. Traditional sequencing techniques have been utilized in order to identify and utilize healthier embryos resulting in a higher success rate. The drawbacks, however, have been that these methods have been far too costly to be within reach of most patients.

Dr Wells and an international research team developed an embryo selection process using much faster and less costly next generation sequencing techniques. This process only takes about 16 hours avoiding the need for embryo freezing. Dr Wells technique also offers additional advantages when compared to previous techniques. Complete chromosome information can be produced and serious gene defects can be detected simultaneously.

As next generation sequencing has been revolutionizing many areas of genetic research, it looks poised to soon benefit IVF patients. “In the past few years, results from randomized clinical trials have suggested that most IVF patients would benefit from embryo chromosome screening, with some studies reporting a 50% boost in pregnancy rates. However, the costs of these genetic tests are relatively high, putting them beyond the reach of many patients. Next generation sequencing is a way which could make chromosome testing more widely available to a greater number of patients, improving access by cutting the costs. Our next step is a randomized clinical trial to reveal the true efficacy of this approach – and this will begin later this year.”  -Dr Wells

5 Next Generation Sequencing Market Drivers and Hindrances

The next generation sequencing market is poised to reach $2.7 billion by 2017 as presented by marketsandmarkets.com. This represents an estimated growth of 16.3% from a market value of $1.3 billion in 2012. Marketsandmarkets presents a detailed analysis of their findings from which the following top next generation sequencing market drivers and hindrances are derived:

5 Top NGS Market Drivers:

  1. Continuous innovations and developments in the market aimed at higher throughput, increased accuracy, and affordable costs
  2. Improved turnaround time
  3. Government funding
  4. Increasing opportunities in NGS clinical applications
  5. Presence of dominant NGS platform players

Although the next generation sequencing market is growing rapidly, there are factors which have been weighing down the market as a whole.

5 Top NGS Market Hindrances:

  1. High reliability on grants and funding from the government
  2. Decrease in capital expenditure from academic institutions
  3. Accuracy and standardization concerns
  4. Data storage problems
  5. Issues in interpretation of complex data

The future of the NGS market promises to offer a simpler, less costly, and more integrated experience for researchers. The market is also poised to revolutionize applied markets like diagnostics, drug discovery, biomarker discovery, personalized medicine, and agriculture and animals research.