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Auto Tag: Targeted region

June 1, 2021  |  

Targeted SMRT Sequencing and phasing using Roche NimbleGen’s SeqCap EZ enrichment

As a cost-effective alternative to whole genome human sequencing, targeted sequencing of specific regions, such as exomes or panels of relevant genes, has become increasingly common. These methods typically include direct PCR amplification of the genomic DNA of interest, or the capture of these targets via probe-based hybridization. Commonly, these approaches are designed to amplify or capture exonic regions and thereby result in amplicons or fragments that are a few hundred base pairs in length, a length that is well-addressed with short-read sequencing technologies. These approaches typically provide very good coverage and can identify SNPs in the targeted region, but are unable to haplotype these variants. Here we describe a targeted sequencing workflow that combines Roche NimbleGen’s SeqCap EZ enrichment technology with Pacific Biosciences’ SMRT Sequencing to provide a more comprehensive view of variants and haplotype information over multi-kilobase regions. While the SeqCap EZ technology is typically used to capture 200 bp fragments, we demonstrate that 6 kb fragments can also be utilized to enrich for long fragments that extend beyond the targeted capture site and well into (and often across) the flanking intronic regions. When combined with the long reads of SMRT Sequencing, multi-kilobase regions of the human genome can be phased and variants detected in exons, introns and intergenic regions.

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June 1, 2021  |  

Minimization of chimera formation and substitution errors in full-length 16S PCR amplification

The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. One popular, efficient method of profiling communities is to amplify and sequence the evolutionarily conserved 16S rRNA sequence. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost and species-level classification is often not possible. SMRT Sequencing easily spans the entire 1.5 kb 16S gene, and in combination with highly-accurate single-molecule sequences, can improve the identification of individual species in a metapopulation. However, when amplifying a mixture of sequences with close similarities, the products may contain chimeras, or recombinant molecules, at rates as high as 20-30%. These PCR artifacts make it difficult to identify novel species, and reduce the amount of productive sequences. We investigated multiple factors that have been hypothesized to contribute to chimera formation, such as template damage, denaturing time before and during cycling, polymerase extension time, and reaction volume. Of the factors tested, we found two major related contributors to chimera formation: the amount of input template into the PCR reaction and the number of PCR cycles. Sequence errors generated during amplification and sequencing can also confound the analysis of complex populations. Circular Consensus Sequencing (CCS) can generate single-molecule reads with >99% accuracy, and the SMRT Analysis software provides filtering of these reads to >99.99% accuracies. Remaining substitution errors in these highly-filtered reads are likely dominated by mis-incorporations during amplification. Therefore, we compared the impact of several commercially-available high-fidelity PCR kits with full-length 16S amplification. We show results of our experiments and describe an optimized protocol for full-length 16S amplification for SMRT Sequencing. These optimizations have broader implications for other applications that use PCR amplification to phase variations across targeted regions and to generate highly accurate reference sequences.

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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.

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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.

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June 1, 2021  |  

Minimization of chimera formation and substitution errors in full-length 16S PCR amplification

The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. Amplification and sequencing of the evolutionarily conserved 16S rRNA gene is an efficient method of profiling communities. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost, and species-level classification is often not possible. PacBio SMRT Sequencing easily spans the entire 1.5 kb 16S gene in a single read, producing highly accurate single-molecule sequences that can improve the identification of individual species in a metapopulation.However, this process still relies upon PCR amplification from a mixture of similar sequences, which may result in chimeras, or recombinant molecules, at rates upwards of 20%. These PCR artifacts make it difficult to identify novel species, and reduce the amount of informative sequences. We investigated multiple factors that may contribute to chimera formation, such as template damage, denaturation time before and during thermocycling, polymerase extension time, and reaction volume. We found two related factors that contribute to chimera formation: the amount of input template into the PCR reaction, and the number of PCR cycles.A second problem that can confound analysis is sequence errors generated during amplification and sequencing. With the updated algorithm for circular consensus sequencing (CCS2), single-molecule reads can be filtered to 99.99% predicted accuracy. Substitution errors in these highly filtered reads may be dominated by mis-incorporations during amplification. Sequence differences in full-length 16S amplicons from several commercial high-fidelity PCR kits were compared.We show results of our experiments and describe our optimized protocol for full-length 16S amplification for SMRT Sequencing. These optimizations have broader implications for other applications that use PCR amplification to phase variations across targeted regions and generate highly accurate reference sequences.

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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.

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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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June 1, 2021  |  

Screening for causative structural variants in neurological disorders using long-read sequencing

Over the past decades neurological disorders have been extensively studied producing a large number of candidate genomic regions and candidate genes. The SNPs identified in these studies rarely represent the true disease-related functional variants. However, more recently a shift in focus from SNPs to larger structural variants has yielded breakthroughs in our understanding of neurological disorders.Here we have developed candidate gene screening methods that combine enrichment of long DNA fragments with long-read sequencing that is optimized for structural variation discovery. We have also developed a novel, amplification-free enrichment technique using the CRISPR/Cas9 system to target genomic regions.We sequenced gDNA and full-length cDNA extracted from the temporal lobe for two Alzheimer’s patients for 35 GWAS candidate genes. The multi-kilobase long reads allowed for phasing across the genes and detection of a broad range of genomic variants including SNPs to multi-kilobase insertions, deletions and inversions. In the full-length cDNA data we detected differential allelic isoform complexity, novel exons as well as transcript isoforms. By combining the gDNA data with full-length isoform characterization allows to build a more comprehensive view of the underlying biological disease mechanisms in Alzheimer’s disease. Using the novel PCR-free CRISPR-Cas9 enrichment method we screened several genes including the hexanucleotide repeat expansion C9ORF72 that is associated with 40% of familiar ALS cases. This method excludes any PCR bias or errors from an otherwise hard to amplify region as well as preserves the basemodication in a single molecule fashion which allows you to capture mosaicism present in the sample.

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June 1, 2021  |  

Targeted enrichment without amplification and SMRT Sequencing of repeat-expansion disease causative genomic regions

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. Using human genomic DNA samples and this strategy, we have successfully targeted the loci of a number of repeat expansion disorders (HTT, FMR1, ATXN10, C9orf72). With this data, we demonstrate the ability to isolate hundreds of individual on-target molecules and accurately sequence through long repeat stretches, regardless of the extreme GC-content, followed by accurate sequencing on a single PacBio RS II SMRT Cell or Sequel SMRT Cell 1M. The method is compatible with multiplexing of multiple targets and multiple samples in a single reaction. Furthermore, this technique also preserves native DNA molecules for sequencing, allowing for the possibility of direct detection and characterization of epigenetic signatures. We demonstrate detection of 5-mC in human promoter sequences and CpG islands.

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June 1, 2021  |  

Amplification-free targeted enrichment and SMRT Sequencing of repeat-expansion genomic regions

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.

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June 1, 2021  |  

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be economical for obtaining sequence information for defined regions of the genome. However, most target enrichment methods are reliant upon some form of amplification which can negatively impact downstream analysis. For example, amplification removes epigenetic marks present in native DNA, including nucleotide methylation, which are hypothesized to contribute to disease mechanisms in some disorders. In addition, some genomic regions known to be causative of many genetic disorders have extreme GC content and/or repetitive sequences that tend to be recalcitrant to faithful amplification. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system to target individual genes. This method, in conjunction with the long reads, high consensus accuracy, and uniform coverage of SMRT Sequencing, allows accurate sequence analysis of complex genomic regions that cannot be investigated with other technologies. Using this strategy, we have successfully targeted a number of repeat expansion disorder loci (HTT, FMR1, ATXN10, C9orf72).With this data, we demonstrate the ability to isolate thousands of individual on-target molecules and, using the Sequel System, accurately sequence through long repeats regardless of the extreme GC-content. The method is compatible with multiplexing of multiple target loci and multiple samples in a single reaction. Furthermore, because there is no amplification step, this technique also preserves native DNA molecules for sequencing, allowing for the direct detection and characterization of epigenetic signatures. To this end, we demonstrate the detection of 5-mC in the CGG repeat of the FMR1 gene that is responsible for Fragile X syndrome.

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June 1, 2021  |  

No-amp targeted SMRT sequencing using a CRISPR-Cas9 enrichment method

Targeted sequencing of genomic DNA requires an enrichment method to generate detectable amounts of sequencing products. Genomic regions with extreme composition bias and repetitive sequences can pose a significant enrichment challenge. Many genetic diseases caused by repeat element expansions are representative of these challenging enrichment targets. PCR amplification, used either alone or in combination with a hybridization capture method, is a common approach for target enrichment. While PCR amplification can be used successfully with genomic regions of moderate to high complexity, it is the low-complexity regions and regions containing repetitive elements sometimes of indeterminate lengths due to repeat expansions that can lead to poor or failed PCR enrichment. We have developed an enrichment method for targeted SMRT Sequencing on the PacBio Sequel System using the CRISPR-Cas9 system that requires no PCR amplification. Briefly, a preformed SMRTbell library containing the target region of interest is cleaved with Cas9 through direct interaction with a sequence-specific guide RNA. After ligation with new poly(A) hairpin adapters, the asymmetric SMRTbell templates are enriched by magnetic bead separation. This method, paired with SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows sequencing of genomic regions regardless of challenging sequence context that cannot be investigated with other technologies. The method is amenable to analyzing multiple samples and/or targets in a single reaction. In addition, this method also preserves epigenetic modifications allowing for the detection and characterization of DNA methylation which has been shown to be a key factor in the disease mechanism for some repeat expansion diseases. Here we present results of our latest No-Amp Targeted Sequencing procedure applied to the characterization of CAG triplet repeat expansions in the HTT gene responsible for Huntington’s Disease.

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April 21, 2020  |  

Long-read sequencing for rare human genetic diseases.

During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is <50% using these approaches, and there remain many rare genetic diseases with unknown cause. There may be many reasons for this, but one plausible explanation is that the responsible mutations are in regions of the genome that are difficult to sequence using conventional technologies (e.g., tandem-repeat expansion or complex chromosomal structural aberrations). Despite the drawbacks of high cost and a shortage of standard analytical methods, several studies have analyzed pathogenic changes in the genome using long-read sequencers. The results of these studies provide hope that further application of long-read sequencers to identify the causative mutations in unsolved genetic diseases may expand our understanding of the human genome and diseases. Such approaches may also be applied to molecular diagnosis and therapeutic strategies for patients with genetic diseases in the future.

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April 21, 2020  |  

Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~?1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

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