Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing

AbstractCombining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the hemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100kb) and MN (~200kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genetic organization and synteny of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes.

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A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2

BMC Genomics ◽

10.1186/s12864-020-07283-6 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Daryl M. Gohl ◽

John Garbe ◽

Patrick Grady ◽

Jerry Daniel ◽

Ray H. B. Watson ◽

...

Keyword(s):

Large Scale ◽

Low Cost ◽

Cost Effective ◽

Clinical Diagnostics ◽

Data Repositories ◽

Coverage Metrics ◽

Sequence Capture ◽

Sequencing Technologies ◽

Public Data ◽

Long Read

Abstract Background The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Results Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach. Conclusions The tailed amplicon method we describe represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

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A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

10.1101/2020.05.11.088724 ◽

2020 ◽

Author(s):

Daryl M. Gohl ◽

John Garbe ◽

Patrick Grady ◽

Jerry Daniel ◽

Ray H. B. Watson ◽

...

Keyword(s):

Large Scale ◽

Low Cost ◽

Cost Effective ◽

Clinical Diagnostics ◽

Data Repositories ◽

Coverage Metrics ◽

Sequence Capture ◽

Sequencing Technologies ◽

Public Data ◽

Long Read

AbstractThe global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach and represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

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Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture

Frontiers in Genetics ◽

10.3389/fgene.2021.743230 ◽

2021 ◽

Vol 12 ◽

Author(s):

Valentina Grosso ◽

Luca Marcolungo ◽

Simone Maestri ◽

Massimiliano Alfano ◽

Denise Lavezzari ◽

...

Keyword(s):

Repeat Expansion ◽

Single Nucleotide ◽

Short Read Sequencing ◽

Sequence Capture ◽

Long Read ◽

Repeat Expansions ◽

Nucleotide Resolution ◽

Generation Sequencing ◽

Single Nucleotide Resolution

Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

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