PacBio SMRT Sequencing
细节
CD Genomics 提供 PacBio SMRT 测序以补充我们的 NGS 设施。通过利用 PacBio 开发的长读长和单分子测序能力，我们很自豪能够提供先进的基因组 从头 组装解决方案和全长基因/转录本测序策略，以满足您的项目需求。
PacBio SMRT测序简介
单分子实时 (SMRT) 测序采用专门的流通池，其中包含数千个带有透明底部的单独皮升孔 - 零模波导 (ZMW)。聚合酶固定在孔的底部，并允许 DNA 链通过 ZMW。因此，该系统可以专注于单个分子。SMRT 测序允许对与单个 DNA 模板分子一起合成的荧光标记核苷酸进行实时成像。当模板和聚合酶解离时，测序反应结束。PacBio 仪器的平均读取长度约为 2 kb，有些读取可能超过 20 kb。更长的读取对于可以跨越更多重复和碱基的新基因组的从头组装特别有用。
在真核和原核基因组中发现的高度重复元件对基因组组装提出了挑战，并使重复序列的详细研究变得困难。长读长测序可提供超过数个或数十个千碱基 (kbs) 的读段，这些读段可以通过单个连续读段跨越复杂或重复的区域，从而解析这些大型结构特征。除了来自单个未扩增分子的相当长且高度准确的 DNA 序列外，它还可以展示甲基化碱基发生的位置，从而提供有关由基因组编码的 DNA 甲基转移酶的功能信息。PacBio SMRT测序在从头基因组学、宏基因组学、转录组学和表观遗传学的研究中具有独特的优势。
PacBio SMRT 测序的优势
- 最长平均读取长度
- 最高的共识准确性
- 统一覆盖
- 同时表观遗传表征
- 单分子分辨率
- 快速且实惠
我们的 PacBio SMRT 测序服务
我们利用先进的 PacBio SMRT 仪器（PacBio SR II 和 PacBio Sequel）用于多种研究目的，包括全基因组从头基因组组装、全长目标测序、宏基因组学研究、全长转录本测序和基因组- 广泛的 DNA 修饰分析。我们经验丰富的专家团队按照每个程序执行质量管理，以确保获得自信和公正的结果。
- 长读长宏基因组测序
- 细菌全基因组从头测序
- 真菌全基因组从头测序
- 全长转录本测序（Iso-Seq）
- 人类基因组 PacBio SMRT 测序
- 全长 16S/18S/ITS rRNA 测序
我们的生物信息学管道包括从头组装、碱基修饰检测、单分子共识生成、转录分析、扩增子分析、序列比对和变异检测。并且可以根据您的特定需求进行更多数据挖掘。
由我们经验丰富的科学家和先进的平台的支持，CD基因组学可以帮助你在研究基因组学，转录组，表观基因组，微生物基因组学和单细胞测序。具有无与伦比的读取长度、均匀的覆盖范围和高精度。如果您有其他要求或问题，请随时与我们联系。
常见问题
RS II系统由PacBio于2013年发布，基于SMRT技术，平均读取长度达到10 kb，最长的超过20 kb。Sequel 是 PacBio 的最新平台，同样基于 SMRT 技术，但在通量和数据质量方面有了很大的提高。
表 1. PacBio RS II 与 Sequel 的比较。
| 参数 | 二级 | 续集 |
| 原则 | SMRT | SMRT |
| 平均读取长度 | 10-15kb | 8-12kb |
| 数据大小/SMRTcells | 500Mb-1Gb (750Mb-1.5Gb) | 5-10Gb |
| 最大输出/运行 | 18Gb (24Gb) | 160Gb |
| 运行时间/SMRTcell | 0.5-6小时 | 0.5-6小时 |
| 多重扩增子 | 384 | 1536 |
2.什么是尺码选择？
PacBio 允许以单分子分辨率对全长 cDNA 文库进行测序。然而，大多数成绩单是 1-1.5kb。由于低丰度、扩增偏差和较小 SMRTbell 结构的优先加载，较长的转录本难以检测。因此，大小选择是显着增加>1.5kb 的转录本数量的有效方法。这对于>3kb 的转录本尤其重要。对于降解的样品，不需要剪切，这可能会进一步减小文库插入片段的大小，并且大小选择对于去除较短的片段很重要，这些片段对组装的好处要少得多。可以使用琼脂糖凝胶或 BluePippin 系统进行大小选择。此外，大小选择可用于全基因组从头测序。
3、DNA样本的要求。
一般而言，提交DNA样本时需要采取以下预防措施：
- 确保 DNA 样本的纯化和完整性。
- 避免 DNA 过度干燥。
- 使用凝胶纯化时，避免基于乙锭/紫外线的可视化。
- 避免过热和涡旋 DNA。
- 进行最少的冻融循环。
- 双链 DNA。
表 2. DNA 样本的估计产量和推荐量。
| 文库插入尺寸 |
推荐提交数量 |
所需的最低浓度（剪切后） |
美东时间。总产量（范围） |
|
|
最小 |
最大限度 |
|||
| 250bp | 600ng | 250ng | 60Gb | 125Gb |
| 500bp | 600ng | 250ng | 10 Gb | 20Gb |
| 1kb | 1.2微克 | 500ng | 90Gb | 180Gb |
| 2kb | 1.2微克 | 500ng | 45Gb | 90Gb |
| 5kb | 2.4微克 | 1微克 | 45Gb | 91Gb |
| 10kb | 2.4微克 | 1微克 | 20Gb | 45Gb |
| 10kb（AMPure 套件） | 10微克 | 5微克 | 90Gb | 182Gb |
| 20kb（AMPure 套件） | 15微克 | 5微克 | 45Gb | 91Gb |
| 20kb（BluePippin 套件） | 15微克 | 5微克 | 9Gb | 18Gb |
4、对RNA样品的要求。
请提供整合和纯化的 RNA 样本（RNA ≥ 5 µg；浓度 ≥ 300 ng/µl）用于全长转录本测序。
参考：
PacBio 的网站
PacBio SMRT Sequencing
- Details
- FAQ
CD Genomics is providing PacBio SMRT sequencing to complement our NGS facility. By taking advantage of the long-read and single molecular sequencing capability developed by PacBio, we are proud to offer advanced genome de novo assembly solutions and full-length gene/transcript sequencing strategy to suit your project needs.
The Introduction of PacBio SMRT Sequencing
Single Molecular Real-Time (SMRT) sequencing employs a specialized flow cell with many thousands of individual picolitre wells with transparent bottoms — zero-mode waveguides (ZMW). The polymerase is fixed to the bottom of the well and allows the DNA strand to progress through the ZMW. As a result, the system can focus on a single molecular. SMRT sequencing allows for real-time imaging of fluorescently tagged nucleotides that are synthesized along with individual DNA template molecules. The sequencing reaction ends when the template and polymerase dissociate. The average read length from the PacBio instrument is approximately 2 kb, and some reads may be over 20 kb. Longer reads are especially useful for de novo assemblies of novel genomes that can span many more repeats and bases.
Highly repetitive elements found in both eukaryotic and prokaryotic genomes pose a challenge for genome assembly and make the detailed study of repetitive sequences difficult. Long-read sequencing delivers reads in excess of several or dozens of kilobases (kbs), which can span complex or repetitive regions with a single continuous read, allowing for the resolution of these large structural features. Besides considerably longer and highly accurate DNA sequences from individual unamplified molecules, it can also exhibit where methylated bases occur, thereby providing functional information about DNA methyltransferases encoded by the genome. PacBio SMRT sequencing has unique advantages in studies of de novo genomics, metagenomics, transcriptomics and epigenetics.
Advantages of PacBio SMRT Sequencing
- Longest average read lengths
- Highest consensus accuracy
- Uniform coverage
- Simultaneous epigenetic characterization
- Single-molecule resolution
- Rapid and affordable
Our PacBio SMRT Sequencing services
We utilize the advanced PacBio SMRT instruments (PacBio SR II and PacBio Sequel) for several research purposes including whole-genome de novo genome assembly, full-length target sequencing, metagenomics studies, full-length transcripts sequencing, and genome-wide DNA modification analysis. Our highly experienced expert team executes quality management following every procedure to ensure confident and unbiased results.
- Long-Read metagenomic sequencing
- Bacterial whole genome de novo sequencing
- Fungal whole genome de novo sequencing
- Full-length transcripts sequencing (Iso-Seq)
- Human genome PacBio SMRT sequencing
- Full-length 16S/18S/ITS rRNA sequencing
Our bioinformatics pipeline includes de novo assembly, base modification detection, single molecule consensus generation, transcript analysis, amplicon analysis, sequence alignment with variant detection. And more data mining are available based on your specific needs.
Supported by our experienced scientists and advanced platforms, CD genomics can assist you in the studies on genomics, transcriptomics, epigenomics, microbial genomics, and single-cell sequencing. with unmatched read lengths, uniform coverage, and high accuracy. If you have additional requirements or questions, please feel free to contact us.
1. What are the differences between PacBio RS II and PacBio Sequel?
RS II system was released by PacBio in 2013, based on the SMRT technology, of which the average read length reaches 10 kb with the longest one beyond 20 kb. Sequel is the newest platform of PacBio that is also based on SMRT technology but has a large increase in throughput and data quality.
Table 1. The comparison between PacBio RS II and Sequel.
| Parameters | RS II | Sequel |
| principles | SMRT | SMRT |
| Average read length | 10-15kb | 8-12kb |
| Data sizes/SMRTcells | 500Mb-1Gb (750Mb-1.5Gb) | 5-10Gb |
| Max Output/Run | 18Gb (24Gb) | 160Gb |
| Run time/SMRTcell | 0.5-6 hours | 0.5-6 hours |
| Multiplex Amplicons | 384 | 1536 |
2. What is size selection?
PacBio allows for the sequencing of full-length cDNA libraries at single-molecule resolution. However, most transcripts are 1-1.5kb. Longer transcripts are hard to detect due to low abundance, amplification bias, and preferential loading of smaller SMRTbell constructs. Therefore, size selection is a powerful way to dramatically increase the number of transcripts>1.5kb. This is especially essential for transcripts>3kb. As for degraded samples, shearing is not necessary, which may further reduce library insert size, and size selection is important to remove shorter fragments that will be much less beneficial in assembly. Size selection can be performed with agarose gels or BluePippin system. Additionally, size selection can be used for whole-genome de novo sequencing.
3. The requirements of DNA samples.
In general, the following precautions need to be taken when submitting DNA samples:
- Make sure the purification and integrity of the DNA samples.
- Avoid overdrying of DNA.
- Avoid ethidium/UV based visualization when using gel purification.
- Avoid overheating and vortexing DNA.
- Undergo a minimum of freeze-thaw cycles.
- Double-stranded DNA.
Table 2. The estimated yield and recommended amounts of DNA samples.
| Library insert size |
Recommended quantity for submission |
Min concentration required (Post-shearing) |
Est. Total yield (range) |
|
|
Min |
Max |
|||
| 250bp | 600ng | 250ng | 60Gb | 125Gb |
| 500bp | 600ng | 250ng | 10 Gb | 20Gb |
| 1kb | 1.2μg | 500ng | 90Gb | 180Gb |
| 2kb | 1.2μg | 500ng | 45Gb | 90Gb |
| 5kb | 2.4μg | 1μg | 45Gb | 91Gb |
| 10kb | 2.4μg | 1μg | 20Gb | 45Gb |
| 10kb (AMPure kit) | 10μg | 5μg | 90Gb | 182Gb |
| 20kb (AMPure kit) | 15μg | 5μg | 45Gb | 91Gb |
| 20kb (BluePippin kit) | 15μg | 5μg | 9Gb | 18Gb |
4. The requirements for RNA samples.
Please provide integrated and purified RNA samples (RNA ≥ 5 µg; concentration ≥ 300 ng/µl) for full-length transcripts sequencing.
Reference:
PacBio’s website
* For Research Use Only. Not for use in diagnostic procedures.
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