De novo assembly is a large part of JGI’s analysis portfolio. Repetitive DNA sequences are abundant in a wide range of organisms we sequence and pose a significant technical challenge for assembly. We are interested in long read technologies capable of spanning genomic repeats to produce better assemblies. We currently have three RS II and two Sequel PacBio machines. RS II machines are primarily used for fungal and microbial genome assembly as well as synthetic biology validation. Between microbes and fungi we produce hundreds of PacBio libraries a year and for throughput reasons the vast majority of these are >10…
Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease. We have developed a…
Most of the base pairs that differ between two human genomes are in intermediate-sized structural variants (50 bp to 5 kb), which are too small to detect with array comparative genomic hybridization or optical mapping but too large to reliably discover with short-read DNA sequencing. Long-read sequencing with PacBio Single Molecule, Real-Time (SMRT) Sequencing platforms fills this technology gap. PacBio SMRT Sequencing detects tens of thousands of structural variants in a human genome with approximately five times the sensitivity of short-read DNA sequencing. Effective application of PacBio SMRT Sequencing to detect structural variants requires quality bioinformatics tools that account for…
Targeted sequencing employing PCR amplification is a fundamental approach to studying human genetic disease. PacBio’s Sequel System and supporting products provide an end-to-end solution for amplicon sequencing, offering better performance to Sanger technology in accuracy, read length, throughput, and breadth of informative data. Sample multiplexing is supported with three barcoding options providing the flexibility to incorporate unique sample identifiers during target amplification or library preparation. Multiplexing is key to realizing the full capacity of the 1 million individual reactions per Sequel SMRT Cell. Two analysis workflows that can generate high-accuracy results support a wide range of amplicon sizes in two…
Animals in the phylum Hemichordata have provided key understanding of the origins and development of body patterning and nervous system organization. However, efforts to sequence and assemble the genomes of highly heterozygous non-model organisms have proven to be difficult with traditional short read approaches. Long repetitive DNA structures, extensive structural variation between haplotypes in polyploid species, and large genome sizes are limiting factors to achieving highly contiguous genome assemblies. Here we present the highly contiguous de novo assembly and preliminary annotation of an indirect developing hemichordate genome, Schizocardium californicum, using SMRT Sequening long reads.
A high quality reference genome is an essential resource for plant and animal breeding and functional and evolutionary studies. The common hop (Humulus lupulus, Cannabaceae) is an economically important crop plant used to flavor and preserve beer. Its genome is large (flow cytometrybased estimates of diploid length >5.4Gb1), highly repetitive, and individual plants display high levels of heterozygosity, which make assembly of an accurate and contiguous reference genome challenging with conventional short-read methods. We present a contig assembly of Cascade Hops using PacBio long reads and the diploid genome assembler, FALCON-Unzip2. The assembly has dramatically improved contiguity and completeness over…
The Pacific Biosciences Iso-Seq method, which can produce high-quality isoform sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. Here, we develop an algorithm called IsoPhase that postprocesses Iso-Seq data to retrieve allele specific isoform information. Using simulated data, we show that for both diploid and tetraploid genomes, IsoPhase results in good SNP recovery with low FDR at error rates consistent with CCS reads. We apply IsoPhase to a haplotyperesolved genome assembly and multiple fetal tissue Iso-Seq dataset from a F1 cross of Angus x Brahman cattle subspecies. IsoPhase-called haplotypes were validated…
Structural variants (genomic differences =50 base pairs) contribute to the evolution of traits and disease. Most structural variants (SVs) are too small to detect with array comparative genomic hybridization and too large to reliably discover with short-read DNA sequencing.
Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).
Jonas Korlach, of PacBio, discusses the use of SMRT sequencing to detect DNA modifications.
Ellen Paxinos, a scientist at PacBio, shares her AGBT poster on work done in collaboration with reference lab Monogram Biosciences using Single Molecule, Real-Time (SMRT) sequencing to detect minor species and variants in HCV. Using two genotypes mixed together, the team was able to detect variants down to 1% and to identify both viral haplotypes from the data. Paxinos says the study is a model for looking at genomic variation in chronic viral infection.
Yunfei Guo, from the University of Southern California, presents his ASHG 2015 poster on a de novo assembly of a diploid Asian genome. The uniform coverage of long-read sequencing helped access regions previously unresolvable due to high GC bias or long repeats. The assembly allowed scientists to fill some 400 gaps in the latest human reference genome, including some as long as 50 kb.
Fritz Sedlazeck, a postdoc at Johns Hopkins University, describes his structural variant detection tool Sniffles in this poster from AGBT 2016. Included: examples of structural variants that could not be detected with other algorithms.
Adam Ameur from the National Genomics Infrastructure at SciLifeLab presented this poster at AGBT 2017. In it, he details a validation study for the use of CRISPR/Cas9 to capture genomic targets without the use of amplification. Results from 12 Huntington’s patients indicate that this approach paired with SMRT Sequencing generates accurate repeat counts in the HTT gene.
Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test that detects the blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection.