Our understanding of human biology and disease is ultimately dependent on a complete understanding of the genome and its functions. The recent application of microarray and sequencing technologies to transcriptomics has changed the simplistic view of transcriptomes to a more complicated view of genome-wide transcription where a large fraction of transcripts emanates from unannotated parts of genomes, and underlined our limited knowledge of the dynamic state of transcription. Most of this broad body of knowledge was obtained indirectly because current transcriptome analysis methods typically require RNA to be converted to complementary DNA (cDNA) before measurements, even though the cDNA synthesis step introduces multiple biases and artefacts that interfere with both the proper characterization and quantification of transcripts. Furthermore, cDNA synthesis is not particularly suitable for the analysis of short, degraded and/or small quantity RNA samples. Here we report direct single molecule RNA sequencing without prior conversion of RNA to cDNA. We applied this technology to sequence femtomole quantities of poly(A)(+) Saccharomyces cerevisiae RNA using a surface coated with poly(dT) oligonucleotides to capture the RNAs at their natural poly(A) tails and initiate sequencing by synthesis. We observed transcript 3' end heterogeneity and polyadenylated small nucleolar RNAs. This study provides a path to high-throughput and low-cost direct RNA sequencing and achieving the ultimate goal of a comprehensive and bias-free understanding of transcriptomes.

We present experimental data concerning potential topological events such as folds, internal backfolds, and/or knots within long molecules of double-stranded DNA when they are stretched by confinement in a nanochannel. Genomic DNA from E. coli was labeled near the ‘GCTCTTC’ sequence with a fluorescently labeled dUTP analog and stained with the DNA intercalator YOYO. Individual long molecules of DNA were then linearized and imaged using methods based on the NanoChannel Array technology (Irys® System) available from BioNano Genomics. Data were collected on 189,153 molecules of length greater than 50 kilobases. A custom code was developed to search for abnormal intensity spikes in the YOYO backbone profile along the length of individual molecules. By correlating the YOYO intensity spikes with the aligned barcode pattern to the reference, we were able to correlate the bright intensity regions of YOYO with abnormal stretching in the molecule, which suggests these events were either a knot or a region of internal backfolding within the DNA. We interpret the results of our experiments involving molecules exceeding 50 kilobases in the context of existing simulation data for relatively short DNA, typically several kilobases. The frequency of these events is lower than the predictions from simulations, while the size of the events is larger than simulation predictions and often exceeds the molecular weight of the simulated molecules. We also identified DNA molecules that exhibit large, single folds as they enter the nanochannels. Overall, topological events occur at a low frequency (~7% of all molecules) and pose an easily surmountable obstacle for the practice of genome mapping in nanochannels.

Here we present a procedure for quantifying single protein molecules affixed to a surface by counting bound antibodies. We systematically investigate many of the parameters that have prevented the robust single-molecule detection of surface-immobilized proteins. We find that a chemically adsorbed bovine serum albumin surface facilitates the efficient detection of single target molecules with fluorescent antibodies, and we show that these antibodies bind for lengths of time sufficient for imaging billions of individual protein molecules. This surface displays a low level of nonspecific protein adsorption so that bound antibodies can be directly counted without employing two-color coincidence detection. We accurately quantify protein abundance by counting bound antibody molecules and perform this robustly in real-world serum samples. The number of antibody molecules we quantify relates linearly to the number of immobilized protein molecules (R(2) = 0.98), and our precision (1-5% CV) facilitates the reliable detection of small changes in abundance (7%). Thus, our procedure allows for single, surface-immobilized protein molecules to be detected with high sensitivity and accurately quantified by counting bound antibody molecules. Promisingly, we can probe flow cells multiple times with antibodies, suggesting that in the future it should be possible to perform multiplexed single-molecule immunoassays.

Using a high-throughput genome mapping approach, we have obtained circa 50 million measurements of the extension of internal human DNA segments in a 41 nm × 41 nm nanochannel. The underlying DNA sequences, obtained by mapping to the reference human genome, are 2.5 to 393 kilobase pairs long and contain % GC contents between 32.5% and 60%. Using Odijk’s theory for a channel-confined wormlike chain, these data reveal that the DNA persistence length increases by almost 20% as the % GC content increases. The increased persistence length is rationalized by a model, containing no adjustable parameters, that treats the DNA as a statistical terpolymer with a sequence-dependent intrinsic persistence length and a sequence-independent electrostatic persistence length.