Robert Morey of Synthetic Genomics discusses how they are using SMRT Sequencing for process development of long, synthetic DNA constructs. PacBio Systems allows them to do larger experiments, cheaper and faster than more traditional approaches, like Sanger Sequencing.
Robert Morey, from Synthetic Genomics shows how his team uses SMRT Sequencing to quickly and accurately confirm the content of long pieces of synthetic DNA. Included: a cost comparison for sequencing clones with Sanger Sequencing vs. SMRT Sequencing.
Benchmark small variant calls are required for developing, optimizing and assessing the performance of sequencing and bioinformatics methods. Here, as part of the Genome in a Bottle (GIAB) Consortium, we apply a reproducible, cloud-based pipeline to integrate multiple short- and linked-read sequencing datasets and provide benchmark calls for human genomes. We generate benchmark calls for one previously analyzed GIAB sample, as well as six genomes from the Personal Genome Project. These new genomes have broad, open consent, making this a ‘first of its kind’ resource that is available to the community for multiple downstream applications. We produce 17% more benchmark…
The advent of Nanopore sequencing has realised portable genomic research and applications. However, state of the art long read aligners and large reference genomes are not compatible with most mobile computing devices due to their high memory requirements. We show how memory requirements can be reduced through parameter optimisation and reference genome partitioning, but highlight the associated limitations and caveats of these approaches. We then demonstrate how these issues can be overcome through an appropriate merging technique. We incorporated multi-index merging into the Minimap2 aligner and demonstrate that long read alignment to the human genome can be performed on a…
Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23…
Single-Molecule Real-Time (SMRT) DNA sequencing is unique in that nucleotide incorporation events are monitored in real time, leading to a wealth of kinetic information in addition to the extraction of the primary DNA sequence. The dynamics of the DNA polymerase that is observed adds an additional dimension of sequence-dependent information, and can be used to learn more about the molecule under study. First, the primary sequence itself can be determined more accurately. The kinetic data can be used to corroborate or overturn consensus calls and even enable calling bases in problematic sequence contexts. Second, using the kinetic information, we can…
In 2012, NIST convened the Genome in a Bottle Consortium to develop the metrology infrastructure needed to enable confidence in human whole genome variant calls.