Mission Bio’s single-cell targeted DNA enrichment by multiplex PCR shows additional capabilities they are developing, and Celsee’s CTC enrichment also claims sc-RNA-Seq and sci-ATAC-seq potential
In 2015 at the AACR conference in Philadelphia PA I noticed a poster that looked eerily familiar – the formation of water-in-oil droplets (“emulsions”) via a microfluidic device, reminiscent of my time in 2009 with then-fledgling RainDance Technologies. This time it was cells in droplets, and the first RainDance presentations had not only DNA but also protein and cells as input materials into the droplet manipulation. The poster presenter was the flow cytometry core facility director at UCSF, and the poster presented the concept (and if memory serves correctly) real-time data for a handful of genes from single cells successfully encapsulated, lysed, and reverse-transcribed inside the individual droplets.
That summer, having just joined SeraCare Life Sciences to define and launch their line of oncology reference standards, I had some extra time in the San Francisco area on a visit there, and visited Dr. Dennis Eastburn in their San Francisco QB3 offices, to ask him about what a single-cell reference material would look like.
As with other technologies, encapsulating cells in droplets is a part of the Mission Bio workflow, but their secret sauce is also in the two-step workflow. This means decoupling the cell and nuclei lysis reaction in one droplet, and then, in a subsequent droplet, combining the cell lysate with cell barcodes, primer pairs and PCR reagents for targeted DNA enrichment. Here’s a September 2018 Genome Research paper that goes into the details.
Four years later Mission Bio hits its stride
Four years later at #AACR19 Mission Bio has not only a key application in longitudinal AML monitoring for research, but also some interesting proof-of-principle posters indicating where they can go with this platform. During their workshop Dr. Nigel Beard their VP of R&D describes the kinds of improvements they are bringing to market: pre-designed amplicons to the ~20,000 genes of the exome, Illumina platform support extending beyond the MiSeq to the HiSeq, NextSeq and NovaSeq, improvements to the amplicon uniformity in representation, and multiplex rising from a current low 100’s to 1000. They also will extend the design reference sequence to mouse, an important application for research.
Their first publication came out in September 2018 titled “High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics”, and one of the authors of that paper, Dr. Koichi Takahashi from MD Anderson Cancer Center in Houston TX presented additional research along the same lines as that paper. He titled his presentation “Unraveling clonal heterogeneity and evolutionary history of AML by single-cell DNA sequencing” and made a clear case of the inferiority of inferring clonal evolution from bulk data compared to directly sequencing and tracking specific mutations from a population of individual cells.
Single-cell DNA analysis for Acute Myeloid Leukemia
In the 2018 paper they presented data from two AML samples, at that time stating in the abstract:
Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.
Dr. Takahashi reported the results of 70 AML patients, the target comprising of 19 AML genes covered by 40 amplicons. All of the reported mutations were confirmed by bulk NGS of the samples. There is some allele dropout (i.e. false homozygote calls due to the alternate allele not being reported) estimated to be 8.7%, and each sample about 270K cells are put into the system, with about 3% of the cells (7617 average) getting individually sequenced.
It was explained later that reproducibility was very high on this system with independent libraries made from additional sets of input cells, even though the sampling rate is low.
Dr. Takahashi had examples of divergent and convergent evolution during the course of the selective pressure of a treatment regimen, and made the point that inferred clonal architecture did not pick up some of the complexities and nuances of being able to follow clonal evolution with populations of single-cell targeted sequencing data.
Single-cell DNA analysis for Chronic Lymphocytic Leukemia
For the second talk, Dr. Esteban Braggio of the Mayo Clinic (Phoenix AZ) gave a talk titled ‘Single-cell genomics studies in MBL and CLL’ (Monoclonal B-cell Lymphocytosis and Chronic Lymphocytic Leukemia). His data were very preliminary – he plans to sequence 500 individual patients but only has data to-date of five samples. He explained that only a very small subset of MBL turns into CLL, and he was using the 500 patient sample-set to look for single-cell genetic clues the progression from MBL to pre-malignant to asymptomatic to progressive states of disease.
Dr. Braggio also demonstrated very good correlation of single-cell variant allele frequency (VAF) compared to the bulk VAF data, accurate down to a measured 1% in the bulk. (Mission Bio states they are accurate down to 0.1%, or one cell in one thousand, which makes sense if you are sequencing 5,000 single cells at a time).
Fascinating Mission Bio possibilities for simultaneous DNA/protein and DNA/RNA analyses
There have been a few single-cell studies for simultaneous DNA and RNA analysis from the same cell. One Mission Bio poster with a collaborator at UCSF showed a proof-of-concept analysis with cells and a pair DNA-labeled antibody (for identification, CD19 and CD30), and clear flow cytometry data that the positive cells for the single antibody were positive, and the negative cells for the single antibody were negative. The workflow was the same as for preparing for flow cytometry, with binding of cells to the antibody and washing, except with single-cell encapsulation, lysing, and analysis of the identification of the antibody via the DNA tag (or no amplification if the antibody is absent). As a proof-of-concept there was only one antibody used, but you can easily imagine the extension of this technology to as many antibodies that you have uniquely barcoded, in CITE-seq fashion.
A second poster from Mission Bio, presented by a former Thermo Fisher colleague of mine Dalia Dhingra, showed the detection of the fusion BCR-ABL transcript (aka the “Philadelphia Chromosome” notable for its frequent presence in CML (chronic myelogenous leukemia) and recently was the first FDA clearance for Bio-Rad’s droplet digital PCR technology). The single cells are encapsulated, lysed, and cDNA is synthesized in the droplet. She was able to demonstrate the presence of both the BCR-ABL transcript in addition to the amplification with variant detection of their standard AML panel within the same cells.
Expect more to come with different kinds of single-cell analysis from this company. It’s nice to see additional choices for customers in the marketplace.
Celsee Inc. surprises with sci-ATAC-Seq
Dr. Swati Ranade (Director of Applications, Celsee) began their workshop with the ‘why’ of single-cell analysis: one primary goal is to understand the heterogeneity of cancer. Through using a gravity-based system, the cells are handled very gently; cells can be visualized in their microwells via microscopy as the bottoms are opaque; and the workflow is semi-automated to get from cells to cDNA for scRNA-seq applications. (Interestingly, at this workshop a scRNA-seq workflow / example was not presented, which was something of a surprise to me as that would be an important large market for them to address, giving 10X Genomics a less expensive alternative.)
One of Dr. Ranade’s slides had an electron micrograph of the hexagonally-shaped ‘open microwell format’ and claimed a >70% recovery of single cells with low doublet rates. I’ve been told they have three microwell formats for scRNA-seq they call CelSingle: a 250K, a 100K and a 50K. With the CelSingle 250K slide, she showed a figure illustrating ~25K cells loaded, the addition of barcoded beads, and the recovery of ~17,500 cells for single-cell transcriptomics, single-cell cytometry, and single-cell proteogenomics applications.
She also showed data of the recovery rate across different number of cells recovered, from 2.5K cells to 22.5K cells, in the 70% to 80% range, and a strong linearity of R^2=0.9997 between cells loaded and cells/beads recovered. Regarding doublets, one slide claimed only 2 doublets observed from 17,500 cells recovered; this is in direct comparison to droplet technology, showing a mouse cell versus human single-cell experiment plotted against each other, where a few dozen doublets were observed.
InCellDx presents on their use of the CelSee technology
Dr. Bruce Patterson, M.D. is the CEO of InCellDx, a single-cell diagnostics company that uses flow cytometry to read out multiplex RNA in-situ hybridization, multiplex protein detection, and DNA/cell cycle genes from tissue and cell suspensions. Their Class I FDA-approved device called incellPREP (IVD/CE-IVD marked) is a non-enzymatic method for producing single-cell suspensions with intact nuclei.
Different types of ASR’s listed on their website indicate the kinds of analytes can be examined via flow cytometry: mRNA probes for CMV or HPV E6 and E7; antibody probes for PD-L1 CD45 or PanCK. Dr. Patterson presented how he uses the CelSee Preparation Slide for CTC enrichment prior to their analyses. (The CelSee Preparation Slide wasn’t laid out in detail; I was told afterwards it has a 30 um screen at the top to catch the large CTCs and let the normal WBCs flow through via gravity, and a small 6 um screen at the bottom to let the liquid flow through.
Single-cell indexing ATAC-Seq from Dr. Andrew Adey at OHSU
Dr. Andrew Adey (OHSU) presentation was entitled “Single-cell omics with microwell miniaturization”, beginning with a popular technique for interrogating open chromatin called ATAC-Seq, which stands for Assay for Transposase-Accessible Chromatin, first described by a group at Stanford in 2013 in Nature Methods. From this 2016 paper in Nature Genetics, Dr. Adey showed a slide of Figure 2 from that paper, with the comment “this figure just about says it all”, the digital signal of regulatory elements almost 2 million annotated regions in number. This method was done in bulk and proved to be a popular method over DNase-I methods used before for looking for open chromatin (areas of active gene expression activity) throughout the genome.
In May of 2015 the single-cell version called sci-ATAC-Seq was first published in Science by Jay Shendure of the University of Washington. The method uses combinatorial indexing, which simply put barcode labels to individual cells in a 96-well plate, pool all the cells together again, and then plate out a second time to about 20 cells per well and index with a separate set of well-specific indices. In March of 2018 the sci-ATAC-Seq method was used in a landmark Drosophila developmental biology paper in the journal Nature titled “The cis-regulatory dynamics of embryonic development at single-cell resolution”. Over 20,000 single nuclei from Drosophila embryos were characterized at three key stages of cell type differentiation.
Dr. Adey showed he was able to successfully apply the sci-ATAC-Seq method to the Celsee platform. He preferred the microwell approach to partitioning, and when I asked him to elaborate on this point later he said the Celsee method had far fewer doublets compared to droplet methods, and that the transparent back of the wells allowed for manual microscopy inspection of the individual wells. Another feature was the open nature of the platform; apparently droplet platforms (namely 10X Genomics although it wasn’t explicitly mentioned) are closed systems not amenable to adjusting different parameters etc.
He applied this technique to a mouse model of Pancreatic Ductal Adenocinoma (abbreviated PDAC), and visualized the sci-ATAC-Seq results using a single-cell RNA-seq t-SNE visualization method developed by Dr. Cole Trapnell called Monocle. (Dr. Trapnell presented a remarkable dataset at #AGBT19 that I still have to digest and write about.) The regulatory elements are pseudo-temporally ordered into something called ‘topics’, or sets of these regulatory elements, shown in the slide below.
He finished his talk opening up a torrent of sci-ATAC methods: for CNVs in single-cells, for methylation, I could not take down notes fast enough for all the applications (at least I could catch two of them). In an endorsement of the Celsee technology, Dr. Adey said ‘it (the barcoding) just worked the first time we tried it’ and they are eager to develop open protocols to share with others.
Afterwards I came by the Celsee booth to take a look at the instrument, where the reagent cassette fits (on the metal rod you can see on top of the manifold in the photo), as well as a photo of one of the CelSingle slides. Well worth looking into – and salespeople reading this, they are hiring (at the moment eight salespeople and two technical specialists) so let them know you read it here first.