Size Matters! Long-Read DNA Sequencing

规模很重要!长readDNA测序

现代基因组学似乎正在经历一个转变，从使用短读技术对大量基因组进行测序，目的是检测snp，到使用长读技术对较少的基因组进行测序，可以解决更复杂的事件，如结构重排、拷贝数变化和重复扩展。

长读测序技术提供了高精度的测序数据，在以下应用领域获得了广泛关注:

长读序列可用于合成长读，以支持从头组装和基因组整理应用。
基因组测序。
长期以来的技术使测序以前具有挑战性的基因组成为可能，
例如，那些有许多高度重复元素的延伸。
基因组定相。
这指的是将等位基因分配到父性和母性染色体上的工作，等位基因是由父性和母性染色体遗传的。
长读测序允许全基因组分阶段研究识别单倍型(共同遗传等位基因)，并重新分阶段
RNA的研究。
长读技术使测序整个转录本和执行直接RNA测序(dRNA-seq)成为可能。
在dRNA-seq中，RNA分子直接测序，不需要修饰，如富集、转化为cDNA或PCR扩增。

因此，dRNA-seq绕过了与RNA处理步骤相关的偏差，同时保留了重要的表观遗传和剪接位点信息。

优质的DNA就是一切!
当我们与长期使用DNA测序的客户交谈时，我们听到的一个挑战是DNA质量。
高质量、完整的高分子量(HMW) DNA是所有长期阅读技术的先决条件。
被认为是HMW的DNA在一定程度上取决于磁场，但一般范围在50 kb到>
300 kb。

Nordic BioSite提供了一套专用的来自Zymo Research的HMW DNA分离试剂盒。
Quick-DNA HMW MagBead试剂盒是一种96孔的格式，可从任何类型的样品(包括生物液体、细胞、固体组织和环境样品)中提取长度可达150 kb的超高浓缩HMW DNA。
分离的DNA可以在第三代测序平台上进行长时间测序，如纳米孔测序和PacBio SMRT测序，无需进一步处理。

请与我们联系，以获得样品，并尝试自己的工具包。

质量控制
鉴于高分子量DNA对长读测序的明显重要性，任何质量控制方案必须提供信息
DNA的完整性以及浓度和纯度。
我们建议使用毛细管电泳，例如生物分析仪，来获得上述质量参数的准确数据。

或者,可以使用相结合的方法来得到一个明确的DNA质量,例如,光谱法来评估浓度和纯度和琼脂糖凝胶电泳评估DNA完整无缺,但定性即涂表明退化但可怜的决议,可能使其难以根据完整性等级样本。

执行长读测序的核心设施和服务提供商通常有输入DNA的质量和浓度阈值
所以一定要提前检查这些，这样你就知道你的样本的目标是什么。

小心轻放!
在提取高质量HMW DNA时，有些样品特别容易，例如血液。
然而，如果处理得当，就有可能从大多数样品类型中获得高质量的起始材料。
请记住，在开始使用任何隔离试剂盒之前，您对样本所做的操作与试剂盒本身一样(如果不是更重要的话)重要。

一般来说，最好从新鲜的材料开始进行长时间测序。
如果不能在采集后立即处理你的样品，正确地储存它们以防止样品中的核酸酶降解是至关重要的。
在液氮中快速冷冻，然后在-80℃保存或保存在合适的稳定缓冲液中，例如，强烈推荐使用Zymo研究中的DNA/RNA屏蔽，以最大限度地减少样品中的DNA降解，直到进行DNA分离和长时间测序。

避免多次冻融循环和任何可能造成混乱和导致不必要的DNA损伤的粗暴处理。

除了样品的储存和保存外，在工作区域中良好的一般处理和护理对处理高分子量DNA或核酸标本大有帮助。
在我们早期的一篇关于成功实现NGS的一般技巧的博客文章中，我们已经详细讨论了这些问题。

取得联系!
在Nordic BioSite，我们相信找到正确的解决方案基于你确切的实验目标和设置。
无论您是在开始长期阅读排序或您正在寻找方法来改善您的工作流程，我们欢迎您通过电子邮件与我们取得联系info@nordicbiosite.com。

Size Matters! Long-Read DNA Sequencing

Modern genomics seems to be undergoing a shift from using short-read technologies to sequence large numbers of genomes with the aim of detecting SNPs, to sequencing fewer genomes using long-read technologies that can resolve more complex events, e.g., structural rearrangements, copy number variations, and repeat expansions.

Long-read sequencing technologies provide highly accurate sequencing data and they are gaining traction within the following application areas:

Long-read sequencing can be used to generate synthetic long reads to support de novo assembly and genome finishing applications.
Genome sequencing. Long-read technologies make it possible to sequence previously challenging genomes, e.g., those with many stretches of highly repetitive elements.
Genome phasing. This refers to the job of assigning alleles to the paternal and maternal chromosomes from which they were inherited. Long-read sequencing permits whole genome phasing studies to identify haplotypes (co-inherited alleles), and to phase de novo
RNA studies. Long-read technologies make it possible to sequence whole transcripts and perform direct RNA sequencing (dRNA-seq). In dRNA-seq, RNA molecules are sequencing directly without the need for modification, e.g., enrichment, conversion to cDNA or PCR amplification. As such, dRNA-seq bypasses biases associated with RNA processing steps, while preserving important epigenetic and splice site information.

Good Quality DNA Is Everything!

One of the challenges we hear about when we speak with customers working with long-read DNA sequencing is DNA quality. High-quality, intact high molecular weight (HMW) DNA is a prerequisite for all long-read technologies. What is considered to be HMW DNA differs somewhat depending on field but in general the range is between 50 kb and > 300 kb.

Nordic BioSite offers a dedicated HMW DNA isolation kit from Zymo Research. The Quick-DNA HMW MagBead Kit is a 96-well format that yields ultra pure and highly concentrated HMW DNA up to 150 kb in length from any sample type, including biological fluids, cells, solid tissue, and environmental samples. Isolated DNA is ready for long-read sequencing on third-generation sequencing platforms such as Nanopore and PacBio SMRT Sequencing without any further processing.

Quality Control

Given the obvious importance of HMW weight DNA to long-read sequencing, any quality control protocol must provide information on DNA intactness as well as concentration and purity. We recommend using capillary electrophoresis e.g., a BioAnalyzer, to obtain accurate data about the aforementioned quality parameters.

Alternatively, it is possible to use a combination of approaches to get a clear picture of DNA quality, e.g., spectrometry to assess concentration and purity and agarose gel electrophoresis to assess DNA intactness, albeit qualitatively i.e. smearing indicates degradation but poor resolution may make it difficult to rank samples according to their intactness.

Core facilities and service providers that perform long-read sequencing usually have quality and concentration thresholds for input DNA so make sure to check with these ahead of time so you know what you are aiming for with your samples.

Handle With Care!

Some sample types are particularly easy when it comes to isolating high-quality HMW DNA, e.g., blood. However, with good handling it should be possible to get high-quality starting material from most sample types. Remember that what you do with your sample before you begin to use any isolation kit is just as (if not more) important as the kit itself.

In general, it is best to start with fresh material for long-read sequencing. If it is not possible to process your samples immediately after collection, it is vital to store them correctly to prevent nucleic acid degradation by nucleases present in the samples. Flash freezing in liquid nitrogen followed by storage at -80 °C or preservation in suitable stabilisation buffers, e.g., DNA/RNA Shield from Zymo Research is highly recommended to minimise DNA degradation in samples until DNA isolation and long-read sequencing is carried out.

Avoid multiple freeze-thaw cycles and any rough handling that may create turbulence and lead to unwanted DNA damage.

Beyond sample storage and preservation, good general handling and care in the working area can go a long way when working with HMW DNA or indeed any nucleic acid specimen. We have covered these in more detail in one of our earlier blog posts about general tips for NGS success.

Get in Touch!

At Nordic BioSite, we believe in finding the right solution based on your exact experimental goals and setup. Whether you are starting out in long-read sequencing or you are looking for ways to improve your workflow, we welcome you to get in touch with us by writing an email to info@nordicbiosite.com.