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Auto Tag: Amplicon

June 1, 2021  |  

Streamlines SMRTbell library generation using addition-only, single tube strategy for all library types reduces time to results

We have streamlined the SMRTbell library generation protocols with improved workflows to deliver seamless end-to-end solutions from sample to analysis. A key improvement is the development of a single-tube reaction strategy that shortened hands-on time needed to generate each SMRTbell library, reduced time-consuming AM Pure purification steps, and minimized sample-handling induced gDNA damage to improve the integrity of long-insert SMRTbell templates for sequencing. The improved protocols support all large-insert genomic libraries, multiplexed microbial genomes, and amplicon sequencing. These advances enable completion of library preparation in less than a day (approximately 4 hours) and opens opportunities for automated library preparation for large-scale projects. Here we share data summarizing performance of the new SMRTbell Express Template Kit 2.0 representing our solutions for 10 kb and >50 kb large-insert genomic libraries, complete microbial genome assemblies, and high-throughput amplicon sequencing. The improved throughput of the Sequel System with read lengths up to 30 kb and high consensus accuracy (> 99.999% accuracy) makes sequencing with high-quality results increasingly assessible to the community.

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June 1, 2021  |  

High-resolution evaluation of gut microbiota associated with intestinal maturation in early preterm neonates

Leaky gut, or intestinal barrier immaturity with elevated intestinal permeability, is the proximate cause of susceptibility to necrotizing enterocolitis in preterm neonates. We recently revealed intestinal barrier maturation was associated with exclusive breastfeeding, less antibiotic exposure, most importantly, altered composition of the gut microbiota. However, sequencing short regions of 16S rRNA gene amplicon failed to identify the specific bacterial groups associated with improved or aberrant intestinal permeability. In this study, we performed high-throughput amplicon sequencing of the full length 16S rRNA gene with single-nucleotide resolution for a cohort of 66 preterm neonates born at 24-33 weeks of gestation who had stool collected daily for 21 postnatal days. We assessed their intestinal permeability by measuring urine non-metabolized sugar probes lactulose and rhamnose during the first 7-10 days of life. We observed that intestinal barrier maturation was positively correlated with changes in specific amplicon sequence variants of species of Clostridiales and Bifidobacterium, while leaky gut was associated with specific strains of Escherichia coli. These results are promising in that they support the use of stool microbial biomarkers for the rapid, non-invasive, and cost-effective assessment of intestinal maturation in neonates.

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June 1, 2021  |  

Microbiome profiling at the strain level using rRNA amplicons

Strain level microbiome profiling is needed for a full understanding of how microbial communities influence human health. Microbiome profiling of rRNA gene amplicons is a well-understood method that is rapid and inexpensive, but standard 16S rRNA gene methods generally cannot differentiate closely related strains. Whole genome/shotgun microbiome profiling is considered a higher-resolution alternative, but with decreased throughput and significantly increased sequencing costs and analysis burden. With both methods there are also challenges with microbial lysis, DNA preparation, and taxonomic analysis. Specialized microbiome-focused protocols were developed to achieve strain-level taxonomic differentiation using a rapid, high throughput rRNA gene assay. The protocol integrates lysis and DNA preparation improvements with a unique high information content amplicon and associated novel database to enable taxonomic differentiation of closely related microbial strains.

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June 1, 2021  |  

The value of long read amplicon sequencing for clinical applications

NGS is commonly used for amplicon sequencing in clinical applications to study genetic disorders and detect disease-causing mutations. This approach can be plagued by limited ability to phase sequence variants and makes interpretation of sequence data difficult when pseudogenes are present. Long-read highly accurate amplicon sequencing can provide very accurate, efficient, high throughput (through multiplexing) sequences from single molecules, with read lengths largely limited by PCR. Data is easy to interpret; phased variants and breakpoints are present within high fidelity individual reads. Here we show SMRT Sequencing of the PMS2 and OPN1 (MW and LW) genes using the Sequel System. Homologous regions make NGS and MLPA results very difficult to interpret.

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June 1, 2021  |  

TLA & long-read sequencing: Efficient targeted sequencing and phasing of the CFTR gene

Background: The sequencing and haplotype phasing of entire gene sequences improves the understanding of the genetic basis of disease and drug response. One example is cystic fibrosis (CF). Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies have revolutionized CF treatment, but only in a minority of CF subjects. Observed heterogeneity in CFTR modulator efficacy is related to the range of CFTR mutations; revertant mutations can modify the response to CFTR modulators, and other intronic variations in the ~200 kb CFTR gene have been linked to disease severity. Heterogeneity in the CFTR gene may also be linked to differential responses to CFTR modulators. The Targeted Locus Amplification (TLA) technology from Cergentis can be used to selectively amplify, sequence and phase the entire CFTR gene. With PacBio long-read SMRT Sequencing, TLA amplicons are sequenced intact and long-range phasing information of all fragments in entire amplicons is retrieved. Experimental Design and Methods: The TLA process produces amplicons consisting of 5-10 proximity ligated DNA fragments. TLA was performed on cell line and genomic DNA from Coriell GM12878, which has few heterozygous SNVs in CFTR, and the IB3 cell line, with known haplotypes but heterozygous for the delta508 mutation. All sample types were prepared with high and low density TLA primer sets, targeting coverage of >100 kb of the CFTR gene. Conclusion: We have demonstrated the power and utility of TLA with long-read SMRT Sequencing as a valuable research tool in sequencing and phasing across very long regions of the human genome. This process can be done in an efficient manner, multiplexing multiple genes and samples per SMRT Cell in a process amenable to high-throughput sequencing.

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June 1, 2021  |  

Comparative metagenome-assembled genome analysis of “Candidatus Lachnocurva vaginae”, formerly known as Bacterial Vaginosis Associated bacterium – 1 (BVAB1)

Bacterial Vaginosis Associated bacterium 1 (BVAB1) is an as-yet uncultured bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales. As its name suggests, this bacterium is often associated with bacterial vaginosis (BV), a common vaginal disorder that has been shown to increase a woman’s risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae infections as well as preterm birth. Further, BVAB1 is associated with the persistence of BV following metronidazole treatment, increased vaginal inflammation, and adverse obstetrics outcomes. There is no available complete genome sequence of BVAB1, which has made it di?cult to mechanistically understand its role in disease. We present here a circularized metagenome-assembled genome (cMAG) of B VAB1 as well as a comparative analysis including an additional six metagenome-assembled genomes (MAGs) of this species. These sequences were derived from cervicovaginal samples of seven separate women. The cMAG is 1.649 Mb in size and encodes 1,578 genes. We propose to rename BVAB1 to “Candidatus Lachnocurva vaginae” based on phylogenetic analyses, and provide genomic evidence that this candidate species may metabolize D-lactate, produce trimethylamine (one of the chemicals responsible for BV-associated odor), and be motile. The cMAG and the six MAGs are valuable resources that will further contribute to our understanding of the heterogeneous etiology of bacterial vaginosis.

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February 5, 2021  |  

Tutorial: SMRT Link overview

This tutorial provides a high-level overview of the features contained within the SMRT Link software. SMRT Link is the web-based end-to-end software workflow manager for run design and set-up on…

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