Circulating tumour DNA analysis demonstrates spatial mutational heterogeneity that coincides with disease relapse in myeloma.

Mutational characterisation in multiple myeloma (MM) currently relies on bone marrow (BM) biopsy, which fails to capture the putative spatial and genetic heterogeneity of this multifocal disease. Analysis of plasma (PL)-derived circulating free tumour DNA (ctDNA) as an adjunct to BM biopsy, for mutational characterisation and tracking disease progression, was evaluated. Paired BM MM cell DNA and ctDNA from 33 relapsed/refractory (RR) and 15 newly diagnosed (ND) patients were analysed for KRAS, NRAS, BRAF and TP53 mutations using the OnTarget Mutation Detection (OMD) platform. OMD detected 128 mutations (PL=31, BM=59, both=38) indicating the presence of PL mutations (54%). A higher frequency of PL-only mutations was detected in RR patients than ND (27.2% vs 6.6%, respectively), authenticating the existence of spatial and genetic heterogeneity in advanced disease. Activating RAS mutations were more highly prevalent than previously described with 69% harboring at least one RAS mutation. Sequential ctDNA quantitation with droplet digital PCR through longitudinal PL tracking of specific clones in seven patients demonstrated changes in fractional abundance of certain clones reflective of the disease status. We conclude that ctDNA analysis as an adjunct to BM biopsy represents a noninvasive and holistic strategy for improved mutational characterisation and therapeutic monitoring of MM.

Oral ulcer by Sphingomonas paucimobilis: first report.

The first case of an oral infection caused by Sphingomonas paucimobilis is reported. A 73-year-old man presented with a gingival ulcer with bone exposure affecting the attached gingiva in the anterior maxillary region. He reported pain during chewing and the presence of fever. Since the first case of S. paucimobilis infection was reported in 1977, involving a leg ulcer, the number of reports related to this organism has been increasing, indicating that the bacterium should be considered an emerging pathogen. It is possible that other non-classical pathogens of the oral cavity may be responsible for infectious lesions, which represents a diagnostic and therapeutic challenge.

Resuspension of DNA sequencing reaction products in agarose increases sequence quality on an automated sequencer.

We are investigating approaches to increase DNA sequencing quality. Since a majorfactor in sequence generation is the cost of reagents and sample preparations, we have developed and optimized methods to sequence directly plasmid DNA isolated from alkaline lysis preparations. These methods remove the costly PCR and post-sequencing purification steps but can result in low sequence quality when using standard resuspension protocols on some sequencing platforms. This work outlines a simple, robust, and inexpensive resuspension protocol for DNA sequencing to correct this shortcoming. Resuspending the sequenced products in agarose before electrophoresis results in a substantial and reproducible increase in sequence quality and read length over resuspension in deionized water and has allowed us to use the aforementioned sample preparation methods to cut considerably the overall sequencing costs without sacrificing sequence quality. We demonstrate that resuspension of unpurified sequence products generated from template DNA isolated by a modified alkaline lysis technique in low concentrations of agarose yields a 384% improvement in sequence quality compared to resuspension in deionized water. Utilizing this protocol, we have produced more than 74,000 high-quality, long-read-length sequences from plasmid DNA template on the MegaBACET 1000 platform.

New DNA sequencing methods.

The Human Genome Project and other major genomic sequencing projects have pushed the development of sequencing technology. In the past six years alone, instrument throughput has increased 15-fold. New technologies are now on the horizon that could yield massive increases in our capacity for de novo DNA sequencing. This review presents a summary of state-of-the-art technologies for genomic sequencing and describes technologies that may be candidates for the next generation of DNA sequencing instruments.

A method for parallel, automated, thermal cycling of submicroliter samples.

A large fraction of the cost of DNA sequencing and other DNA-analysis processes results from the reagent costs incurred during cycle sequencing or PCR. In particular, the high cost of the enzymes and dyes used in these processes often results in thermal cycling costs exceeding $0.50 per sample. In the case of high-throughput DNA sequencing, this is a significant and unnecessary expense. Improved detection efficiency of new sequencing instrumentation allows the reaction volumes for cycle sequencing to be scaled down to one-tenth of presently used volumes, resulting in at least a 10-fold decrease in the cost of this process. However, commercially available thermal cyclers and automated reaction setup devices have inherent design limitations which make handling volumes of <1 microL extremely difficult. In this paper, we describe a method for thermal cycling aimed at reliable, automated cycling of submicroliter reaction volumes.

An automated sample preparation system for large-scale DNA sequencing.

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

An arrayable flow-through microcentrifuge for high-throughput instrumentation.

A compact, flow-through centrifugation system has been developed specifically for high-throughput centrifugation of large numbers of samples. The instrument is based on multiple high-speed rotors that also serve as sample holders. The small size of the rotors allows them to be arrayed in a standard 96-well microtiter plate spacing, making this device ideal for highly parallel automated instrumentation. Though initially designed for cell separation in DNA sequencing protocols, the flow-through centrifuge can be used to replace conventional centrifugation in most processes involving small samples. Techniques for recovery of both the supernatant and the pellet have been developed, as well as techniques for sample mixing, and cleaning of the reusable rotors. This paper discusses the design and performance of the flow-through centrifuge applied to cell separation and resuspension and to DNA purification and concentration.