RESUMO
Long-read sequencing has proven the necessity for high-quality genomic assemblies of reference species, including enigmatic ctenophores. Obtaining high-molecular-weight genomic DNA is pivotal to this process and has proven highly problematic for many species. Here, we discuss different methodologies for gDNA isolation and present a protocol for isolating gDNA for several members of the phylum Ctenophora. Specifically, we describe a Pacific Biosciences library construction method used in conjunction with gDNA isolation methods that have proven successful in obtaining high-quality genomic assemblies in ctenophores.
Assuntos
Ctenóforos , DNA , Genômica , Análise de Sequência de DNA , Animais , Ctenóforos/genética , Genômica/métodos , DNA/genética , DNA/isolamento & purificação , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biblioteca Gênica , Genoma/genéticaRESUMO
Like most other DNA sequencing core facilities, one of our continuing goals is to improve our sequence output without substantially adding to cost. To minimize sample-to-sample variability in template DNA concentration, we implemented the rolling circle amplification (RCA) procedure for preparing our DNA templates. In addition to saving time and reducing the number of steps in template DNA preparation, the RCA method has the potential to normalize the DNA concentration in samples that can be sequenced directly without additional purification. In the present study, we used RCA-generated templates to test a recently reported procedure that increased sequence quality by resuspending the sequenced products in low concentrations of agarose before capillary electrophoresis (CE) on a MegaBACE 1000 platform. Although we did not obtain the expected result using the specified procedure, a modification resulted in up to 60% increase in total sequence yield per sample plate. A combination of agarose and formamide-EDTA in the resuspension solution enabled us to generate long-read and high-quality sequences for more than 38,000 templates with minimal additional cost.
Assuntos
DNA Bacteriano/genética , Eletroforese Capilar/métodos , Análise de Sequência de DNA/métodos , DNA Bacteriano/isolamento & purificação , DNA Circular/genética , Ácido Edético , Eletroforese em Gel de Ágar , Eletroforese Capilar/instrumentação , Desenho de Equipamento , Formamidas , Técnicas de Amplificação de Ácido Nucleico , Plasmídeos , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soluções , Moldes GenéticosRESUMO
We report the complete genome sequence of Carnobacterium gilichinskyi strain WN1359, previously isolated from Siberian permafrost and capable of growth under cold (0°C), anoxic, CO2-dominated, low-pressure (0.7-kPa) conditions in a simulation of the Mars atmosphere.
RESUMO
We report the complete genome sequence of Serratia liquefaciens strain ATCC 27592, which was previously identified as capable of growth under low-pressure conditions. To the best of our knowledge, this is the first announcement of the complete genome sequence of an S. liquefaciens strain.