Genome sequencing of large numbers of individuals promises to advance the understanding, treatment, and prevention of human diseases, among other applications. We describe a genome sequencing platform that achieves efficient imaging and low reagent consumption with combinatorial probe anchor ligation chemistry to independently assay each base from patterned nanoarrays of self-assembling DNA nanoballs. We sequenced three human genomes with this platform, generating an average of 45- to 87-fold coverage per genome and identifying 3.2 to 4.5 million sequence variants per genome. Validation of one genome data set demonstrates a sequence accuracy of about 1 false variant per 100 kilobases. The high accuracy, affordable cost of $4400 for sequencing consumables, and scalability of this platform enable complete human genome sequencing for the detection of rare variants in large-scale genetic studies.
Small non-coding RNAs (sncRNAs) are pervasive regulators of physiological and pathological processes. We previously developed the human miRNA Tissue Atlas, detailing the expression of miRNAs across organs in the human body. Here, we present an updated resource containing sequencing data of 188 tissue samples comprising 21 organ types retrieved from six humans. Sampling the organs from the same bodies minimizes intra-individual variability and facilitates the making of a precise high-resolution body map of the non-coding transcriptome. The data allow shedding light on the organ- and organ system-specificity of piwi-interacting RNAs (piRNAs), transfer RNAs (tRNAs), microRNAs (miRNAs) and other non-coding RNAs. As use case of our resource, we describe the identification of highly specific ncRNAs in different organs. The update also contains 58 samples from six tissues of the Tabula Muris collection, allowing to check if the tissue specificity is evolutionary conserved between Homo sapiens and Mus musculus. The updated resource of 87 252 non-coding RNAs from nine non-coding RNA classes for all organs and organ systems is available online without any restrictions (https://www.ccb.uni-saarland.de/tissueatlas2).
Many detection methods have been used or reported for the diagnosis and/or surveillance of SARS-CoV-2. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive, claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by RT-PCR, probably due to loss or degradation of virus RNA in the sampling process, or even mutation of the virus genome. Therefore, developing highly sensitive methods is imperative to ensure robust detection capabilities. With the goal of improving sensitivity and accommodate various application settings, we developed a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. In addition, optional sequencing can provide further confirmation as well as phylogenetic information of the identified virus(es) for specific strain discrimination, which will be of paramount importance for surveillance purposes that represent a global health imperative. Finally, we also developed in parallel a multiplex-PCR-based metagenomic method that is amenable to detect SARS-CoV-2, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.
Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.
Massively parallel sequencing (MPS) on DNA nanoarrays provides billions of reads at relatively low cost and enables a multitude of genomic applications. Further improvement in read length, sequence quality and cost reduction will enable more affordable and accurate comprehensive health monitoring tests. Currently the most efficient MPS uses dye-labeled reversibly terminated nucleotides (RTs) that are expensive to make and challenging to incorporate. Furthermore, a part of the dye-linker (scar) remains on the nucleobase after cleavage and interferes with subsequent sequencing cycles. We describe here the development of a novel MPS chemistry (CoolMPS TM ) utilizing unlabeled RTs and four natural nucleobasespecific fluorescently labeled antibodies with fast (30 sec) binding. We implemented CoolMPS TM on MGI's PCR-free DNBSEQ MPS platform using arrays of 200nm DNA nanoballs (DNBs) generated by rolling circle replication and demonstrate 3-fold improvement in signal intensity and elimination of scar interference. Single-end 100-400 base and pair-end 2x150 base reads with high quality were readily generated with low out-of-phase incorporation. Furthermore, DNBs with less than 50 template copies were successfully sequenced by strong-signal CoolMPS TM with 3-times higher accuracy than in standard MPS. CoolMPS TM chemistry based on natural nucleobases has potential to provide longer, more accurate and less expensive MPS reads, including highly accurate "4-color sequencing" on the most efficient dyecrosstalk-free 2-color imagers with an estimated sequencing error rate of 0.00058% (one error in 170,000 base calls) in a proof-of-concept demonstration.
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