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Thermo Fisher Working on Direct Library Prep From Samples, 600 Base Reads for Ion Technology

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NEW YORK (GenomeWeb) – Thermo Fisher Scientific is working on several improvements to its Ion Torrent sequencing technology that it plans to roll out later this year, including a method for direct library preparation from biological samples, as well as 600 base reads and one-hour sequencing runs on the Ion S5 and S5 XL platforms, which the company launched last September.

Earlier this month at the Advances in Genome Biology and Technology meeting in Orlando, Florida, Simon Cawley, senior director of software, informatics, and molecular biology for the Ion Torrent platform at Thermo, talked about the new developments during a company workshop, and Thermo scientists provided more details in poster presentations at the company's exhibit booth.

All sequencing platforms to date take purified DNA as input material for their libraries, but Thermo researchers have now developed a method that allows them to generate libraries directly from biological samples, including FFPE sections, blood, saliva, and buccal swabs, without the need to extract the DNA. This significantly reduces the overall time from sample to data analysis and only requires small amounts of sample.

To prepare the samples, the researchers incubated them for five minutes with the firm's so-called "Direct Reagent", the composition of which remains undisclosed, prior to loading them onto the Ion Chef for library prep, using an AmpliSeq gene panel and an AmpliSeq protocol with minor modifications, and clonal amplification. They then sequenced the libraries on either the Ion PGM or the Ion S5 and found that the sequencing performance was similar to purified DNA. Of note, they were able to sequence libraries from single FFPE slices as small as 2 mm2 and single microliters of blood or saliva samples.

Cawley said that for FFPE sequencing, the direct protocol eliminates the need for tissue deparaffinization, protease digestion, and DNA purification. An evaluation of 20 previously characterized cancer FFPE samples showed that the direct approach could detect all previously reported mutations, including single nucleotide variants, deletions, and copy number variants.

Thermo's team has also been increasing the read length on the Ion S5 to more than 600 bases. At the moment, the system offers read lengths up to 400 bases on two of its chips, the Ion 520 and the Ion 530. Longer reads would be advantageous for certain sequencing applications, for example HLA typing and immune repertoire sequencing.

To increase read length, the scientists developed new beads to accommodate longer template DNA molecules, as well as a new long-read sequencing polymerase with improved signal and phase. They also wrote new instrument scripts to conserve reagents and allow for more reagent flows.

To test these developments, they performed HLA typing on 96 samples using DNA fragmented to 600 to 700 base pairs and the 530 chip. They obtained a read-length peak of 612 bases, a mean read length of 549 bases, and a raw read accuracy of 99.5 percent.

In addition, Thermo researchers have developed a rapid sequencing protocol for the Ion S5 XL sequencer, which decreases the total workflow from input DNA to sequence data to as little as 6.5 hours, or a standard workday. This could have advantages for time-sensitive applications such as rapid infectious disease identification, antibiotic susceptibility testing, and preimplantation aneuploidy screening, for which the researchers tested their speed protocol.

To achieve the greater speed, they reduced the time required for each of the sequencing reagent flows on the Ion S5 and worked out the minimum number of flows needed to cover the length of the amplicons in a library. For an AmpliSeq panel to identify bacterial species, for example, they were able to speed up the sequencing run to between 55 and 70 minutes, using 200 or 250 flows.

Together with 3.5 hours of library preparation, 2 hours of isothermal amplification, and 50 to 85 minutes of data analysis, the total workflow came to 6.5 to 8 hours.

Asked by an audience member whether the company has made improvements to the homopolymer indel error rate of the Ion Torrent technology, Cawley said that they have made "significant advances" but that indel error will always remain. He said the technology obtains 99.5 percent accuracy, with the remaining errors concentrated in longer homopolymer regions.

The Ion Torrent technology remains the only commercialized semiconductor sequencing technology to date, though Illumina has said it is working on its own CMOS sequencing technology, dubbed Firefly, which is scheduled to come out in the second half of 2017. "If you don't want to wait a couple of years, come talk to us," Cawley quipped.    

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