John H. Leamon

Genome sequencing in microfabricated high-density picolitre reactors

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

An integrated semiconductor device enabling non-optical genome sequencing

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

The development and impact of 454 sequencing

The 454 Sequencer has dramatically increased the volume of sequencing conducted by the scientific community and expanded the range of problems that can be addressed by the direct readouts of DNA sequence. Key breakthroughs in the development of the 454 sequencing platform included higher throughput, simplified all in vitro sample preparation and the miniaturization of sequencing chemistries, enabling massively parallel sequencing reactions to be carried out at a scale and cost not previously possible. Together with other recently released next-generation technologies, the 454 platform has started to democratize sequencing, providing individual laboratories with access to capacities that rival those previously found only at a handful of large sequencing centers. Over the past 18 months, 454 sequencing has led to a better understanding of the structure of the human genome, allowed the first non-Sanger sequence of an individual human and opened up new approaches to identify small RNAs. To make next-generation technologies more widely accessible, they must become easier to use and less costly. In the longer term, the principles established by 454 sequencing might reduce cost further, potentially enabling personalized genomics.

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies. NOTE: In the version of this article initially published, it should have been acknowledged that Jan F. Simons, in addition to Roman K. Thomas and Elizabeth Nickerson, contributed equally to this work. The error has been corrected in the HTML and PDF versions of the article.

Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing

BackgroundWhole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls.ResultsAll amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR.ConclusionOf the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.

Combining Highly Multiplexed PCR with Semiconductor-Based Sequencing for Rapid Cancer Genotyping

There is growing demand for routine identification of actionable mutations in clinical cancer specimens. Genotyping platforms must provide rapid turnaround times and work effectively with limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissue specimens that often yield poor quality DNA. We describe semiconductor-based sequencing of DNA from FFPE specimens using a single-tube, multiplexed panel of 190 amplicons targeting 46 cancer genes. With just 10 ng of input DNA, average read depths of 2000× can be obtained in 48 hours, with >95% of the reads on target. A validation set of 45 FFPE tumor specimens containing 53 point mutations previously identified with a mass spectrometry-based genotyping platform, along with 19 indels ranging from 4 to 63 bp, was used to evaluate assay performance. With a mutant allele ratio cutoff of 8%, we were able to achieve 100% sensitivity (95% CI = 97.3% to 100.0%) and 95.1% specificity (95% CI = 91.8% to 98.0%) of point mutation detection. All indels were visible by manual inspection of aligned reads; 6/9 indels ≤12 bp long were detected by the variant caller software either exactly or as mismatched nucleotides within the indel region. The rapid turnaround time and low input DNA requirements make the multiplex PCR and semiconductor-based sequencing approach a viable option for mutation detection in a clinical laboratory.

Overview: methods and applications for droplet compartmentalization of biology

Three protocols in this issue highlight applications of emulsification procedures, which deliver high-throughput potential to the molecular biology laboratory, without the need for automation. These procedures have already generated interesting results and spurred the development of exciting new technologies, while requiring only readily available laboratory equipment.

Bias in Whole Genome Amplification: Causes and Considerations.

Whole genome amplification (WGA) is a widely used molecular technique that is becoming increasingly necessary in genetic research on a range of sample types including individual cells, fossilized remains and entire ecosystems. Multiple methods of WGA have been developed, each with specific strengths and weaknesses, but with a common defect in that each method distorts the initial template DNA during the course of amplification. The type, extent, and circumstance of the bias vary with the WGA method and particulars of the template DNA. In this review, we endeavor to discuss the types of bias introduced, the susceptibility of common WGA techniques to these bias types, and the interdependence between bias and characteristics of the template DNA. Finally, we attempt to illustrate some of the criteria specific to the analytical platform and research application that should be considered to enable combination of the appropriate WGA method, template DNA, sequencing platform, and intended use for optimal results.

HIGH-THROUGHPUT, MASSIVELY PARALLEL DNA SEQUENCING TECHNOLOGY FOR THE ERA OF PERSONALIZED MEDICINE

A challenge to the realization of the vision of personalized medicine is our lack of understanding of the amount of genetic variation contained in the human species, which has been prohibitively expensive to acquire to date. In this review, we discuss a new system based on massively parallel picotiter plate based sequencing. This approach, now commercially available, delivers in excess of 200,000 individual clonal reads in a single five hour run and hence provides orders of magnitude improvement in speed and cost over conventional Sanger-based sequencing. The system is scalable well beyond its current performance and its emergence makes plausible the routine sequencing of individual human genomes in the near future. In this review, our system is presented in light of recent NHGRI sequencing technology grants, and a number of new applications enabled by ready access to hundreds of thousands of individual clonal reads are discussed with a particular focus on their potential impact on human health.