从解剖千足虫中分离组织特异性DNA用于纳米孔测序。

IF 1.3 Q3 BIOCHEMICAL RESEARCH METHODS
Biology Methods and Protocols Pub Date : 2025-05-28 eCollection Date: 2025-01-01 DOI:10.1093/biomethods/bpaf042
Elena Cruz, William Wittstock, Bruce A Snyder, Arnab Sengupta
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引用次数: 0

摘要

在双足纲中大约有12000种已被描述的物种。在16个已描述目中的4个目中,只有5个物种完成了基因组测序。没有完整的基因组可以用于像木蝇科这样难以置信的多样化家庭。此外,与这些物种的关键功能有关的遗传信息非常有限。人们对表征非模式生物的基因组越来越感兴趣,然而,为具有复杂形态的生物体提取高质量的DNA可能具有挑战性。在这里,我们描述了一种详细的方法,从腿部、头部和身体组织中获得高纯度的DNA,这些DNA来自野生捕获的千足虫物种Cherokia georgiana。我们的解剖方案分离消化道,最大限度地减少提取DNA样本中的微生物丰度。我们描述了样品均质步骤,提高总DNA产量。为了评估样品的质量、浓度和大小,我们分别使用分光光度法、荧光法和自动电泳法。我们一直得到平均DNA长度在12-25 kb以上。我们采用了Oxford Nanopore Technologies的MinION长读测序技术,这是一种价格合理且易于使用的选择,具有现场应用的潜力。在这里,我们提出了组织特异性DNA测序指标,线粒体DNA一致序列的比对和组装,以及系统发育分析。虽然注意到我们基于纳米孔的测序方法的局限性,但我们提供了一个框架来处理现场标本,以获得可用于基因特异性比对和分析的无pcr DNA测序数据。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Tissue-specific DNA isolation from dissected millipedes for nanopore sequencing.

There are approximately 12,000 described species within the class Diplopoda. Only five species, falling within 4 of 16 described orders, have fully sequenced genomes. No whole genomes are available for incredibly diverse families like Xystodesmidae. Furthermore, genetic information attributed to key functions in these species is very limited. There is a growing interest in characterizing genomes of non-model organisms, however, extracting high-quality DNA for organisms with complex morphology can be challenging. Here we describe a detailed methodology for obtaining high-purity DNA from legs, head, and body tissues from wild-caught specimens of the millipede species Cherokia georgiana. Our dissection protocol separates the digestive tract minimizing microbial abundance in the extracted DNA sample. We describe sample homogenization steps that improve total DNA yield. To assess sample quality, concentration, and size we use spectrophotometry, fluorometry, and automated electrophoresis, respectively. We consistently obtain average DNA length upwards of 12-25 kb. We applied Oxford Nanopore Technologies MinION long-read sequencing, an affordable and accessible option with potential for field-based applications. Here we present tissue-specific DNA sequencing metrics, alignment and assembly of mitochondrial DNA consensus sequence, and phylogenetic analysis. While noting the limitations of our nanopore-based sequencing methodology, we provide a framework to process field specimens for PCR-free DNA sequencing data that can be used for gene-specific alignment and analysis.

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来源期刊
Biology Methods and Protocols
Biology Methods and Protocols Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)
CiteScore
3.80
自引率
2.80%
发文量
28
审稿时长
19 weeks
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