June 1, 2021  |  

Data release for polymorphic genome assembly algorithm development.

Heterozygous and highly polymorphic diploid (2n) and higher polyploidy (n > 2) genomes have proven to be very difficult to assemble. One key to the successful assembly and phasing of polymorphic genomics is the very long read length (9-40 kb) provided by the PacBio RS II system. We recently released software and methods that facilitate the assembly and phasing of genomes with ploidy levels equal to or greater than 2n. In an effort to collaborate and spur on algorithm development for assembly and phasing of heterozygous polymorphic genomes, we have recently released sequencing datasets that can be used to test and develop highly polymorphic diploid and polyploidy assembly and phasing algorithms. These data sets include multiple species and ecotypes of Arabidopsis that can be combined to create synthetic in-silico F1 hybrids with varying levels of heterozygosity. Because the sequence of each individual line was generated independently, the data set provides a ‘ground truth’ answer for the expected results allowing the evaluation of assembly algorithms. The sequencing data, assembly of inbred and in-silico heterozygous samples (n=>2) and phasing statistics will be presented. The raw and processed data has been made available to aid other groups in the development of phasing and assembly algorithms.


June 1, 2021  |  

Progress on the reassembly and annotation of the goat genome.

The goat (Capra hircus) remains an important livestock species due to the species’ ability to forage and provide milk, meat and wool in arid environments. The current goat reference assembly and annotation borrows heavily from other loosely related livestock species, such as cattle, and may not reflect the unique structural and functional characteristics of the species. We present preliminary data from a new de novo reference assembly for goat that primarily utilizes 38 million PacBio P5-C3 reads generated from an inbred San Clemente goat. This assembly consists of only 5,902 contigs with a contig N50 size of 2.56 megabases which were grouped into scaffolds using cis-chromosome associations generated by the analysis of Hi-C sequence reads. To provide accurate functional genetic annotation, we utilized existing RNA-seq data and generated new data consisting of over 784 million reads from a combination of 27 different developmental timepoints/tissues. This dataset provides a tangible improvement over existing goat genomics resources by correcting over 247 misassemblies in the current goat reference genome and by annotating predicted gene models with actual expressed transcript data. Our goal is to provide a high quality resource to researchers to enable future genomic selection and functional prediction within the field of goat genomics.


June 1, 2021  |  

Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human diseases.

The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans. Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building complete transcript isoforms has been very challenging. We have developed the Iso-Seq technique, which is capable of sequencing full-length, single-molecule cDNA sequences. The method employs SMRT Sequencing to generate individual molecules with average read lengths of more than 10 kb and some as long as 40 kb. As most transcripts are from 1 to 10 kb, we can sequence through entire RNA molecules, requiring no fragmentation or post-sequencing assembly. Jointly with the sequencing method, we developed a computational pipeline that polishes these full-length transcript sequences into high-quality, non-redundant transcript consensus sequences. Iso-Seq sequencing enables unambiguous identification of alternative splicing events, alternative transcriptional start and poly-A sites, and transcripts from gene fusion events. Knowledge of the complete set of isoforms from a sample of interest is key for accurate quantification of isoform abundance when using any technology for transcriptome studies. Here we characterize the full-length transcriptome of normal human tissues, paired tumor/normal samples from breast cancer, and a brain sample from a patient with Alzheimer’s using deep Iso-Seq sequencing. We highlight numerous discoveries of novel alternatively spliced isoforms, gene-fusions events, and previously unannotated genes that will improve our understanding of human diseases.


June 1, 2021  |  

Full-length env deep sequencing in a donor with broadly neutralizing V1/V2 antibodies.

Background: Understanding the co-evolution of HIV populations and broadly neutralizing antibody (bNAb) lineages may inform vaccine design. Novel long-read, next-generation sequencing methods allow, for the first time, full-length deep sequencing of HIV env populations. Methods: We longitudinally examined env populations (12 time points) in a subtype A infected individual from the IAVI primary infection cohort (Protocol C) who developed bNAbs (62% ID50>50 on a diverse panel of 105 viruses) targeting the V1/V2 region. We developed a Pacific Biosciences single molecule, real-time sequencing protocol to deeply sequence full-length env from HIV RNA. Bioinformatics tools were developed to align env sequences, infer phylogenies, and interrogate escape dynamics of key residues and glycosylation sites. PacBio env sequences were compared to env sequences generated through amplification and cloning. Env dynamics were interpreted in the context of the development of a V1/V2-targeting bNAb lineage isolated from the donor. Results: We collected a median of 6799 high quality full-length env sequences per timepoint (median per-base accuracy of 99.7%). A phylogeny inferred with PacBio and 100 cloned env sequences (10 time points) found cloned env sequences evenly distributed among PacBio sequences. Phylogenetic analyses also revealed a potential transient intra-clade superinfection visible as a minority variant (~5%) at 9 months post-infection (MPI), and peaking in prevalence at 12MPI (~64%), just preceding the development of heterologous neutralization. Viral escape from the bNAb lineage was evident at V2 positions 160, 166, 167, 169 and 181 (HxB2 numbering), exhibiting several distinct escape pathways by 40MPI. Conclusions: Our PacBio full-length env sequencing method allowed unprecedented characterization of env dynamics and revealed an intra-clade superinfection that was not detected through conventional methods. The importance of superinfection in the development of this donor’s V1/V2-directed bNAb lineage is under investigation. Longitudinal full-length env deep sequencing allows accurate phylogenetic inference, provides a detailed picture of escape dynamics in epitope regions, and can identify minority variants, all of which may prove useful for understanding how env evolution can drive the development of antibody breadth.


June 1, 2021  |  

Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human cancer

The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans (1). Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building complete transcript isoforms has been very challenging (2). We have developed a technique, called Iso-Seq sequencing, that is capable of sequencing full-length, single-molecule cDNA sequences. The method employs SMRT Sequencing from PacBio, which can sequence individual molecules with read lengths that average more than 10 kb and can reach as long as 40 kb. As most transcripts are from 1 – 10 kb, we can sequence through entire RNA molecules, requiring no fragmentation or post-sequencing assembly. Jointly with the sequencing method, we developed a computational pipeline that polishes these full-length transcript sequences into high-quality, non-redundant transcript consensus sequences. Iso-Seq sequencing enables unambiguous identification of alternative splicing events, alternative transcriptional start and polyA sites, and transcripts from gene fusion events. Knowledge of the complete set of isoforms from a sample of interest is key for accurate quantification of isoform abundance when using any technology for transcriptome studies (3). Here we characterize the full-length transcriptome of paired tumor/normal samples from breast cancer using deep Iso-Seq sequencing. We highlight numerous discoveries of novel alternatively spliced isoforms, gene-fusion events, and previously unannotated genes that will improve our understanding of human cancer. (1) Faustino NA and Cooper TA. Genes and Development. 2003. 17: 419-437(2) Steijger T, et al. Nat Methods. 2013 Dec;10(12):1177-84.(3) Au KF, et al. Proc Natl Acad Sci U S A. 2013 Dec 10;110(50):E4821-30.


June 1, 2021  |  

Targeted sequencing of genes from soybean using NimbleGen SeqCap EZ and PacBio SMRT Sequencing

Full-length gene capture solutions offer opportunities to screen and characterize structural variations and genetic diversity to understand key traits in plants and animals. Through a combined Roche NimbleGen probe capture and SMRT Sequencing strategy, we demonstrate the capability to resolve complex gene structures often observed in plant defense and developmental genes spanning multiple kilobases. The custom panel includes members of the WRKY plant-defense-signaling family, members of the NB-LRR disease-resistance family, and developmental genes important for flowering. The presence of repetitive structures and low-complexity regions makes short-read sequencing of these genes difficult, yet this approach allows researchers to obtain complete sequences for unambiguous resolution of gene models. This strategy has been applied to genomic DNA samples from soybean coupled with barcoding for multiplexing.


June 1, 2021  |  

A comprehensive study of the sugar pine (Pinus lambertiana) transcriptome implemented through diverse next-generation sequencing approaches

The assembly, annotation, and characterization of the sugar pine (Pinus lambertiana Dougl.) transcriptome represents an opportunity to study the genetic mechanisms underlying resistance to the invasive white pine blister rust (Cronartium ribicola) as well as responses to other abiotic stresses. The assembled transcripts also provide a resource to improve the genome assembly. We selected a diverse set of tissues allowing the first comprehensive evaluation of the sugar pine gene space. We have combined short read sequencing technologies (Illumina MiSeq and HiSeq) with the relatively new Pacific Biosciences Iso-Seq approach. From the 2.5 billion and 1.6 million Illumina and PacBio (46 SMRT cells) reads, 33,720 unigenes were de novo assembled. Comparison of sequencing technologies revealed improved coverage with Illumina HiSeq reads and better splice variant detection with PacBio Iso-Seq reads. The genes identified as unique to each library ranges from 199 transcripts (basket seedling) to 3,482 transcripts (female cones). In total, 10,026 transcripts were shared by all libraries. Genes differentially expressed in response to these provided insight on abiotic and biotic stress responses. To analyze orthologous sequences, we compared the translated sequences against 19 plant species, identifying 7,229 transcripts that clustered uniquely among the conifers. We have generated here a high quality transcriptome from one WPBR susceptible and one WPBR resistant sugar pine individual. Through the comprehensive tissue sampling and the depth of the sequencing achieved, detailed information on disease resistance can be further examined.


June 1, 2021  |  

Alternative splicing in FMR1 premutations carriers

Over 40% of males and ~16% of female carriers of a FMR1 premutation allele (55-200 CGG repeats) are at risk for developing Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), an adult onset neurodegenerative disorder while, about 20% of female carriers will develop Fragile X-associated Primary Ovarian Insufficiency (FXPOI), in addition to a number of adult-onset clinical problems (FMR1 associated disorders). Marked elevation in FMR1 mRNA levels have been observed with premutation alleles and the resulting RNA toxicity is believed to be the leading molecular mechanism proposed for these disorders. The FMR1 gene, as many housekeeping genes, undergoes alternative splicing. Using long-read isoform sequencing (SMRT) and qRT-PCR we have recently reported that, although the relative abundance of all FMR1 mRNA isoforms is significantly increased in the premutation group compared to controls, there is a disproportionate increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14. In total, we confirmed the existence of 16 out of 24 predicted isoforms in our samples. However, it is unknown, which isoforms, when overexpressed, may contribute to the premutation pathology. To address this question we have further defined the transcriptional FMR1 isoforms distribution pattern in different tissues, including heart, muscle, brain and testis derived from FXTAS premutation carriers and age-matched controls. Preliminary data indicates the presence of a transcriptional signature of the FMR1 gene, which clusters more by individual than by tissue type. We identified additional isoforms than the 16 reported in our previous study, including a group with particular splice patterns that were observed only in premutations but not in controls. Our findings suggest that the characterization of expression levels of the different FMR1 isoforms is fundamental for understanding the regulation of the FMR1 gene as well as for elucidating the mechanism(s) by which “toxic gain of function” of the FMR1 mRNA may play a role in FXTAS and/or in the other FMR1-associated conditions. In addition to the elevated levels of FMR1 isoforms, the altered abundance/ratio of the corresponding FMRP isomers may affect the overall function of FMRP in premutations.


June 1, 2021  |  

De novo assembly and preliminary annotation of the Schizocardium californicum genome

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.


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