Scientists at Münchner Leukämielabor, a large leukemia diagnostic laboratory in Germany, have developed a gene panel to analyze acute myeloid leukemia and myelodysplastic syndromes using RainDance's single-droplet PCR and Illumina's MiSeq.
The panel, which covers almost 30 genes and was developed by MLL in collaboration with RainDance, is currently being validated and is expected to enter clinical use next month. It will complement MLL's other sequencing-based assays, which run on the 454 GS FLX and GS Junior as well as on Sanger sequencing instrumentation.
For the last two years, MLL has been routinely sequencing panels of a few genes on the 454, both as diagnostic markers and to detect minimal residual disease in leukemia patients (CSN 2/15/2012).
In collaboration with 454, MLL also developed two primer sets to analyze leukemia genes on the 454 platforms, which Roche sells for research use only (CSN 4/25/2012).
But because the number of molecular markers in myeloid disease, such as AML or myelodysplastic syndromes, continues to increase, the company decided to develop a larger panel with RainDance, according to Alexander Kohlmann, who heads MLL's next-gen sequencing and microarray department.
MLL had already tested the RainDance RDT 1000 system earlier this year, generating a small proof-of-principle library to amplify five genes for sequencing on the 454. For the larger myeloid panel, they decided to combine RainDance's single-droplet PCR with the MiSeq because the number of amplicons would be too large for the 454 and sequencing costs too high.
The researchers initially also considered Illumina's TruSeq assay, which allows for multiplex amplification, but decided to use RainDance's single-plex assay instead. According to Kohlmann, amplification with RainDance is more homogeneous, and it is easier to add new content to an existing library.
The length of the amplicons is currently about 170 base pairs, which MLL sequences bidirectionally on the MiSeq, using 2x160 base paired-end reads.
With the expected MiSeq upgrade later this year, the RainDance library will be modified to increase the amplicon size and take "full advantage" of the increased read length of 2x250 base pairs, Kohlmann said.
Longer reads will be particularly useful to detect large duplications and complicated insertions and deletions, he said. "That's clearly one of the drawbacks of the shorter read lengths, in comparison to the 454."
As part of an ongoing validation and verification study, MLL has already studied approximately 60 previously analyzed patient samples using the RainDance/MiSeq combo, including samples with complex insertions and duplications.
To set up the droplet-based PCR reactions, MLL maintains a RainDance RDT 1000 and recently acquired the Thunderstorm, which has a higher throughput. Four patient samples are combined on each MiSeq run and are sequenced to a target coverage of 2,000-fold per gene. So far, MLL has been able to detect clones with a frequency as low as 5 percent and indels of up to 27 base pairs.
Starting next month, MLL plans to migrate some patients with myeloid malignancies onto the new RainDance/MiSeq workflow. Later this year, the company also wants to generate performance data on the assay's sensitivity, specificity, and linearity for detecting mutation levels so the test can be accredited by the German national accreditation body, DAkkS.
The new assay includes some genes that MLL currently analyzes by 454, and several that it sequences by Sanger because they contain homopolymers. "We see promising performance now of these genes on the MiSeq, so we will migrate a few genes from the 454 and also a few genes from Sanger sequencing into this new panel and create some extra space on the 454 for other cases," Kohlmann said.
The turnaround time of the new panel is about equal to analyzing the same genes by 454. "From start to finish, it will not be a big change for us having those genes sequenced on the 454 or using the RainDance workflow and the MiSeq," he said. However, an advantage of a larger panel is that all results are available at the same time.
Besides the myeloid panel, MLL may develop two additional RainDance libraries for MiSeq analysis: a smaller panel for lymphocytic leukemia and a larger research cancer panel.
But Kohlmann said that for the time being, the MiSeq will complement rather than replace the 454 at MLL. "There will always be high-throughput genes that we need to sequence on the 454," he said, and an important GC-rich gene, CEBPA, is still sequenced best on the 454 platform.
In the meantime, he and his colleagues are "still actively scouting what's left and right of 454 and Illumina," including semiconductor and nanopore sequencing. However, "we need to make sure that what is placed in our lab works today because we need to accommodate requests for daily routine diagnostics," he said. "That's why 454 is still our workhorse."