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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  |  

Long read sequencing technology to solve complex genomic regions assembly in plants

Numerous whole genome sequencing projects already achieved or ongoing have highlighted the fact that obtaining a high quality genome sequence is necessary to address comparative genomics questions such as structural variations among genotypes and gain or loss of specific function. Despite the spectacular progress that has been done regarding sequencing technologies, accurate and reliable data are still challenging, at the whole genome scale but also when targeting specific genomic regions. These issues are even more noticeable for complex plant genomes. Most plant genomes are known to be particularly challenging due to their size, high density of repetitive elements and various levels of ploidy. To overcome these issues, we have developed a strategy in order to reduce the genome complexity by using the large insert BAC libraries combined with next generation sequencing technologies. We have compared two different technologies (Roche-454 and Pacific Biosciences PacBio RS II) to sequence pools of BAC clones in order to obtain the best quality sequence. We targeted nine BAC clones from different species (maize, wheat, strawberry, barley, sugarcane and sunflower) known to be complex in terms of sequence assembly. We sequenced the pools of the nine BAC clones with both technologies. We have compared results of assembly and highlighted differences due to the sequencing technologies used. We demonstrated that the long reads obtained with the PacBio RS II technology enables to obtain a better and more reliable assembly notably by preventing errors due to duplicated or repetitive sequences in the same region.


June 1, 2021  |  

Low-input long-read sequencing for complete microbial genomes and metagenomic community analysis

Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. The resulting library can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes using the SMRT Portal assembly program HGAP, plus base modification analysis. The entire process can be done in less than 3 days by standard laboratory personnel. This approach is particularly important for analysis of metagenomic communities, in which genomic DNA is often limited. From these samples, full-length 16S amplicons can be generated, prepped with the standard SMRTbell library prep protocol, and sequenced. Alternatively, a 2 kb sheared library, made from a few ng of input DNA, can also be used to elucidate the microbial composition of a community, and may provide information about biochemical pathways present in the sample. In both these cases, 1-2 kb reads with >99.9% accuracy can be obtained from Circular Consensus Sequencing.


June 1, 2021  |  

Application specific barcoding strategies for SMRT Sequencing

Over the last few years, several advances were implemented in the PacBio RS II System to maximize throughput and efficiency while reducing the cost per sample. The number of useable bases per SMRT Cell now exceeds 1 Gb with the latest P6-C4 chemistry and 6-hour movies. For applications such as microbial sequencing, targeted sequencing, Iso-Seq (full-length isoform sequencing) and Nimblegen’s target enrichment method, current SMRT Cell yields could be an excess relative to project requirements. To this end, barcoding is a viable option for multiplexing samples. For microbial sequencing, multiplexing can be accomplished by tagging sheared genomic DNA during library construction with modified SMRTbell adapters. We studied the performance of 2- to 8-plex microbial sequencing. For full-length amplicon sequencing such as HLA typing, amplicons as large as 5 kb may be barcoded during amplification using barcoded locus-specific primers. Alternatively, amplicons may be barcoded during SMRTbell library construction using barcoded SMRTbell adapters. The preferred barcoding strategy depends on the user’s existing workflow and flexibility to changing and/or updating existing workflows. Using barcoded adapters, five Class I and II genes (3.3 – 5.8 kb) x 96 patients can be multiplexed and typed. For Iso-Seq full-length cDNA sequencing, barcodes are incorporated during 1st-strand synthesis and are enabled by tailing the oligo-dT primer with any PacBio published 16-bp barcode sequences. RNA samples from 6 maize tissues were multiplexed to generate barcoded cDNA libraries. The NimbleGen SeqCap Target Enrichment method, combined with PacBio’s long-read sequencing, provides comprehensive view of multi-kilobase contiguous regions, both exonic and intronic regions. To make this cost effective, we recommend barcoding samples for pooling prior to target enrichment and capture. Here, we present specific examples of strategies and best practices for multiplexing samples for different applications for SMRT Sequencing. Additionally, we describe recommendations for analyzing barcoded samples.


June 1, 2021  |  

Multiplexing strategies for microbial whole genome SMRT Sequencing

The increased throughput of the RS II and Sequel Systems enables multiple microbes to be sequenced on a single SMRT Cell. This multiplexing can be readily achieved by simply incorporating a unique barcode for each microbe into the SMRTbell adapters after shearing genomic DNA using a streamlined library construction process. Incorporating a barcode without the requirement for PCR amplification prevents the loss of epigenetic information (e.g., methylation signatures), and the generation of chimeric sequences, while the modified protocol eliminates the need to build several individual SMRTbell libraries. We multiplexed up to 8 unique strains of H. pylori. Each strain was sheared, and processed through adapter ligation in a single, addition only reaction. The barcoded strains were then pooled in equimolar quantities, and processed through the remainder of the library preparation and purification steps. We demonstrate successful de novo microbial assembly and epigenetic analysis from all multiplexes (2 through 8-plex) using standard tools within SMRT Link Analysis using data generated from a single SMRTbell library, run on a single SMRT Cell. This process facilitates the sequencing of multiple microbial genomes in a single day, greatly increasing throughput and reducing costs per genome assembly.


June 1, 2021  |  

Enrichment of unamplified DNA and long-read SMRT Sequencing in unlocking the underlying biological disease mechanisms of repeat expansion disorders

For many of the repeat expansion disorders, the disease gene has been discovered, however the underlying biological mechanisms have not yet been fully understood. This is mainly due to technological limitations that do not allow for the needed base-pair resolution of the long, repetitive genomic regions. We have developed a novel, amplification-free enrichment technique that uses the CRISPR/Cas9 system to target large repeat expansions. This method, in conjunction with PacBio’s long reads and uniform coverage, enables sequencing of these complex genomic regions. By using a PCR-free amplification method, we are able to access not only the repetitive elements and interruption sequences accurately, but also the epigenetic information.


June 1, 2021  |  

Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing

Next-Generation Sequencing (NGS) technologies allow for molecular profiling of cancer samples with high sensitivity and speed at reduced cost. For efficient profiling of cancer samples, it is important that the NGS methods used are not only robust, but capable of accurately detecting low-frequency somatic mutations. Single Molecule, Real-Time (SMRT) Sequencing offers several advantages, including the ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach. The availability of genetically defined, human genomic reference standards provides an industry standard for the development and quality control of molecular assays for studying cancer variants. Here we characterize SMRT Sequencing for the detection of low-frequency somatic variants using the Quantitative Multiplex DNA Reference Standards from Horizon Discovery, combined with amplification of the variants using the Multiplicom Tumor Hotspot MASTR Plus assay. First, we sequenced a reference standard containing precise allelic frequencies from 1% to 24.5% for major oncology targets verified using digital PCR. This reference material recapitulates the complexity of tumor composition and serves as a well-characterized control. The control sample was amplified using the Multiplicom Tumor Hotspot MASTR Plus assay that targets 252 amplicons (121-254 bp) from 26 relevant cancer genes, which includes all 11 variants in the control sample. Next, we sequenced control samples prepared by SeraCare Life Sciences, which contained a defined mutation at allelic frequencies from 10% down to 0.1%. The wild type and mutant amplicons were serially diluted, sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants.


June 1, 2021  |  

Candidate gene screening using long-read sequencing

We have developed several candidate gene screening applications for both Neuromuscular and Neurological disorders. The power behind these applications comes from the use of long-read sequencing. It allows us to access previously unresolvable and even unsequencable genomic regions. SMRT Sequencing offers uniform coverage, a lack of sequence context bias, and very high accuracy. In addition, it is also possible to directly detect epigenetic signatures and characterize full-length gene transcripts through assembly-free isoform sequencing. In addition to calling the bases, SMRT Sequencing uses the kinetic information from each nucleotide to distinguish between modified and native bases.


June 1, 2021  |  

Application-specific barcoding strategies for SMRT Sequencing

The increased sequencing throughput creates a need for multiplexing for several applications. We are here detailing different barcoding strategies for microbial sequencing, targeted sequencing, Iso-Seq full-length isoform sequencing, and Roche NimbleGen’s target enrichment method.


June 1, 2021  |  

Multiplexing strategies for microbial whole genome SMRT Sequencing

As the throughput of the PacBio Systems continues to increase, so has the desire to fully utilize SMRT Cell sequencing capacity to multiplex microbes for whole genome sequencing. Multiplexing is readily achieved by incorporating a unique barcode for each microbe into the SMRTbell adapters and using a streamlined library preparation process. Incorporating barcodes without PCR amplification prevents the loss of epigenetic information and the generation of chimeric sequences, while eliminating the need to generate separate SMRTbell libraries. We multiplexed the genomes of up to 8 unique strains of H. pylori. Each genome was sheared and processed through adapter ligation in a single, addition-only reaction. The barcoded samples were pooled in equimolar quantities and a single SMRTbell library was prepared. We demonstrate successful de novo microbial assembly from all multiplexes tested (2- through 8-plex) using data generated from a single SMRTbell library, run on a single SMRT Cell with the PacBio RS II, and analyzed with standard SMRT Analysis assembly methods. This strategy was successful using both small (1.6 Mb, H. pylori) and medium (5 Mb, E. coli) genomes. This protocol facilitates the sequencing of multiple microbial genomes in a single run, greatly increasing throughput and reducing costs per genome.


June 1, 2021  |  

Characterization of the Poly-T variants in the TOMM40 gene using PacBio long reads

Genes associated with several neurological disorders have been shown to be highly polymorphic. Targeted sequencing of these genes using NGS technologies is a powerful way to increase the cost-effectiveness of variant discovery and detection. However, for a comprehensive view of these target genes, it is necessary to have complete and uniform coverage across regions of interest. Unfortunately, short-read sequencing technologies are not ideal for these types of studies as they are prone to mis-mapping and often fail to span repetitive regions. Targeted sequencing with PacBio long reads provides the unique advantage of single-molecule observations of complex genomic regions. PacBio long reads not only provide continuous sequence data though polymorphic or repetitive regions, but also have no GC bias. Here we describe the characterization of the poly-T locus in TOMM40, a gene known to be associated with progression to Alzheimer’s, using PacBio long reads. Probes were designed to capture a 20 kb region comprising the TOMM40 and ApoE genes. Target regions were captured in multiple cell lines and sequencing libraries made using standard sample preparation methods. We will present our results on the poly-T structural variants that we observed in TOMM40 in these cell lines. We will also present our results on probe design optimization and barcoding strategies for a cost-effective solution.


June 1, 2021  |  

Enrichment of unamplified DNA and long-read SMRT Sequencing to unlock repeat expansion disorders

Nucleotide repeat expansions are a major cause of neurological and neuromuscular disease in humans, however, the nature of these genomic regions makes characterizing them extremely challenging. Accurate DNA sequencing of repeat expansions using short-read sequencing technologies is difficult, as short-read technologies often cannot read through regions of low sequence complexity. Additionally, these short reads do not span the entire region of interest and therefore sequence assembly is required. Lastly, most target enrichment methods are reliant upon amplification which adds the additional caveat of PCR bias. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system for specific targeting of individual human genes. This method, in conjunction with PacBio’s long reads and uniform coverage, enables sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples and this strategy, we have successfully targeted the loci of Huntington’s Disease (HTT; CAG repeat), Fragile X (FMR1; CGG repeat), ALS (C9orf72; GGGGCC repeat), and Spinocerebellar ataxia type 10 (SCA10; variable ATTCT repeat) for examination. With this data, we demonstrate the ability to isolate hundreds of individual on-target molecules in a single SMRT Cell and accurately sequence through long repeat stretches, regardless of the extreme GC-content. The method is compatible with multiplexing of multiple targets and multiple samples in a single reaction. This technique also captures native DNA molecules for sequencing, allowing for the possibility of direct detection and characterization of epigenetic signatures.


June 1, 2021  |  

Target enrichment using a neurology panel for 12 barcoded genomic DNA samples on the PacBio SMRT Sequencing platform

Target enrichment is a powerful tool for studies involved in understanding polymorphic SNPs with phasing, tandem repeats, and structural variations. With increasing availability of reference genomes, researchers can easily design a cost-effective targeted investigation with custom probes specific to regions of interest. Using PacBio long-read technology in conjunction with probe capture, we were able to sequence multi-kilobase enriched regions to fully investigate intronic and exonic regions, distinguish haplotypes, and characterize structural variations. Furthermore, we demonstrate this approach is advantageous for studying complex genomic regions previously inaccessible through other sequencing platforms. In the present work, 12 barcoded genomic DNA (gDNA) samples were sheared to 6 kb for target enrichment analysis using the Neurology panel provided by Roche NimbleGen. Probe-captured DNA was used to make SMRTbell libraries for SMRT Sequencing on the PacBio RS II. Our results demonstrate the ability to multiplex 12 samples and achieve 1300x enrichment of targeted regions. In addition, we achieved an even representation of on-target rate of 70% across the 12 barcoded genomic DNA samples.


June 1, 2021  |  

“SMRTer Confirmation”: Scalable clinical read-through variant confirmation using the Pacific Biosciences SMRT Sequencing platform

Next-generation sequencing (NGS) has significantly improved the cost and turnaround time for diagnostic genetic tests. ACMG recommends variant confirmation by an orthogonal method, unless sufficiently high sensitivity and specificity can be demonstrated using NGS alone. Most NGS laboratories make extensive use of Sanger sequencing for secondary confirmation of single nucleotide variants (SNVs) and indels, representing a large fraction of the cost and time required to deliver high quality genetic testing data to clinicians and patients. Despite its established data quality, Sanger is not a high-throughput method by today’s standards from either an assay or analysis standpoint as it can involve manual review of Sanger traces and is not amenable to multiplexing. Toward a scalable solution for confirmation, Invitae has developed a fully automated and LIMS-tracked assay and informatics pipeline that utilizes the Pacific Biosciences SMRT sequencing platform. Invitae’s pipeline generates PCR amplicons that encompass the variant(s) of interest, which are converted to closed DNA structures (SMRTbells) and sequenced in pools of 96 per SMRTcell. Each amplicon is appended with a 16nt barcode that encodes the patient and variant IDs. Per-sample de-multiplexing, alignment, variant calling, and confirmation resolution are handled via an automated pipeline. The confirmation process was validated by analyzing 243 clinical SNVs and indels in parallel with the gold standard Sanger sequencing method. Amplicons were sequenced and analyzed in technical replicates to demonstrate reproducibility. In this study, the PacBio-based confirmation pipeline demonstrated high reproducibility (97.5%), and outperformed Sanger in the fraction of primary NGS variants confirmed (PacBio = 93.4% and 94.7% confirmed across two replicates, Sanger = 84.8%) while having 100% concordance of confirmation status among overlapping confirmation calls.


June 1, 2021  |  

Targeted SMRT Sequencing of difficult regions of the genome using a Cas9, non-amplification based method

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 novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system for specific targeting of individual human genes. This method, in conjunction with SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows the sequencing of complex genomic regions that cannot be investigated with other technologies.


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