NGS 试剂盒

是，它们是相同的磁珠，仅灌装量不同。

对于组合和独特的双标签接头，使用以下序列来进行接头修剪：读数 1：AGATCGGAAGAGCACACGTCTGAACTCCAGTCA读数 2：AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

Twist Bioscience 保证试剂盒有效期自发货之日起至少为 3 个月。

安全数据表可从我们的网页中获取，点击此处下载。

请注意，我们的网页上仅提供了 NGS 产品的 SDS 文件；如需获得我们合成生物学产品的 SDS 文件，请发送电子邮件至 customersupport@twistbioscience.com 联系我们。

是，Twist 接头与酶切或机械片段化所产生的 DNA 片段相容。

是，Twist 文库制备试剂盒适合制作用于全基因组测序的文库。

由于大多数 WGS 应用都使用较长的读取长度（全基因组测序为 2×151，而相比之下全外显子组测序为 2×101 或 2×76），因此需要改变片段化条件，以适应更长的片段长度。

对于酶切片段化试剂盒，长度取决于三个因素：时间、温度和 DNA 输入

对于机械片段化，应对条件（时间、功率、占空比和每个脉冲的周期）进行设置，以适应更长的片段化长度。

可以，Twist 提供灵活的 NGS 试剂盒组件购买选择。您可以登录您的 Twist 账户，获取可订购组件的完整列表。您可以订购以下组件：

文库制备组件 

• PN 100253 – TWIST 文库制备 EF 试剂盒 1，16 个样本    
• PN 100875 – TWIST 文库制备 MF 试剂盒 1，16 个样本   
• PN 100401 – TWIST 文库制备试剂盒 2，16 个样本                     
• PN 100572 – TWIST 文库制备 EF 试剂盒 1，96 个样本    
• PN 100876 – TWIST 文库制备 MF 试剂盒 1，96 个样本   
• PN 100573 – TWIST 文库制备试剂盒 2，96 个样本                     

组合接头    

• PN 100255 – 16 个样本 

唯一双标签接头         

• PN 101307 – TWIST 通用接头系统，16 个样本    
• PN 101308 – TWIST 通用接头系统，96 个样本 - 孔板 A           
• PN 101309 – TWIST 通用接头系统，96 个样本 - 孔板 B
• PN 101310 – TWIST 通用接头系统，96 个样本 - 孔板 C
• PN 101311 – TWIST 通用接头系统，96 个样本 - 孔板 D

封闭剂      

• PN 100856 – TWIST 通用封闭剂，2 次反应   
• PN 100578 – TWIST 通用封闭剂，12 次反应 
• PN 100767 – TWIST 通用封闭剂，96 次反应             

* 杂交试剂         

• PN 101263 – 杂交试剂，2 次反应      
• PN 100930 – 杂交试剂，12 次反应    
• PN 100982 – 杂交试剂，96 次反应    

* 快速杂交试剂  

• PN 101260 – 快速杂交试剂，2 次反应         
• PN 100964 – 快速杂交试剂，12 次反应       
• PN 100968 – 快速杂交试剂，96 次反应       

* 清洗缓冲液 

• PN 101264 – 清洗缓冲液，2 次反应 
• PN 100985 – 清洗缓冲液，12 次反应          
• PN 100846 – 清洗缓冲液，96 次反应          

* 快速清洗缓冲液      

• PN 101261 – 快速清洗缓冲液，2 次反应    
• PN 100971 – 快速清洗缓冲液，12 次反应  
• PN 100972 – 快速清洗缓冲液，96 次反应  

纯化与结合磁珠          

• PN 101262 – 纯化与结合磁珠，2 次反应      
• PN 100983 – 纯化与结合磁珠，12 次反应    
• PN 100984 – 纯化与结合磁珠，96 次反应   

* 注：标准杂交试剂仅能与标准清洗缓冲液一起使用；相反，快速杂交试剂仅能与快速清洗缓冲液一起使用。若将标准杂交试剂混入快速清洗缓冲液（以及将快速杂交试剂混入标准清洗缓冲液）会导致性能不佳。

是的，除了机械片段化试剂盒外，Twist 还提供以下与酶切片段化一起使用的文库制备试剂盒：

100253：Twist 文库制备试剂盒，酶切片段化，16 个样本（包括酶和缓冲液）

101059：Twist 文库制备试剂盒，酶切片段化，16 个样本（包括酶、缓冲液和纯化磁珠）

100572：Twist 文库制备试剂盒，酶切片段化，96 个样本（包括酶和缓冲液）

101058：Twist 文库制备试剂盒，酶切片段化，96 个样本（包括酶、缓冲液和纯化磁珠）

注意，您还需要以下接头才能进行文库制备：

101307：Twist 通用接头系统，16 个样本

101308：Twist 通用接头系统，96 个样本——孔板 A

101309：Twist 通用接头系统，96 个样本——孔板 B

101310：Twist 通用接头系统，96 个样本——孔板 C

101311：Twist 通用接头系统，96 个样本——孔板 D

是的，除了酶切片段化试剂盒外，Twist 还提供以下与机械片段化一起使用的文库制备试剂盒：

100875：Twist 文库制备试剂盒，机械片段化，16 个样本（包括酶和缓冲液）

101280：Twist 文库制备试剂盒，机械片段化，16 个样本（包括酶、缓冲液和纯化磁珠）

100876：Twist 文库制备试剂盒，机械片段化，96 个样本（包括酶和缓冲液）

101281：Twist 文库制备试剂盒，机械片段化，96 个样本（包括酶、缓冲液和纯化磁珠）

注意，您还需要以下接头才能进行文库制备：

101307：Twist 通用接头系统，16 个样本

101308：Twist 通用接头系统，96 个样本——孔板 A

101309：Twist 通用接头系统，96 个样本——孔板 B

101310：Twist 通用接头系统，96 个样本——孔板 C

101311：Twist 通用接头系统，96 个样本——孔板 D

推荐平均靶向片段大小为 200 bp；如此一来，平均文库大小约为 350 bp；然而，您应该根据测序读长对插入大小进行调整。

Twist 针对酶切片段化和机械片段化提供了完整*试剂盒——请注意这些试剂盒不包括富集组合：

酶切片段化            

• PN 101897 – TWIST EF 文库制备 + UDI 和标准杂交，16×2 次反应          
• PN 101896 – TWIST EF 文库制备 + UDI 和快速杂交，16×2 次反应     
• PN 101901 – TWIST EF 文库制备 + UDI 和标准杂交，96×12 次反应       
• PN 101900 – TWIST EF 文库制备 + UDI 和快速杂交，96×12 次反应    

机械片段化  

• PN 101899 – TWIST MF 文库制备 + UDI 和标准杂交，16×2 次反应       
• PN 101898 – TWIST MF 文库制备 + UDI 和快速杂交，16×2 次反应    
• PN 101903 – TWIST MF 文库制备 + UDI 和标准杂交，96×12 次反应     
• PN 101902 – TWIST MF 文库制备 + UDI 和快速杂交，96×12 次反应

*注：要生成和扩增文库，您必须购买 KAPA® HiFi HotStart ReadyMix (Kapa Biosystems®, #KK2601) 或其同等产品。

假设 DNA 输入量为 50 ng，则接头与 DNA 的比例大约为 200:1。

The TrueAmp Library Preparation Kit is designed for flexible DNA library construction across a wide range of sample types and input amounts. It supports an integrated workflow where fragmentation, end repair, and dA-tailing are performed in a single enzyme mix, simplifying the overall process.

The kit performs robustly across diverse DNA inputs, including FFPE samples or samples with varying GC content and is compatible with DNA in common storage buffers such as TE, Tris-HCl, or water.

It also enables consistent and reliable fragmentation using a single standardized protocol, while also allowing users to tune insert size by adjusting incubation time or temperature. The streamlined workflow can be completed in approximately 3 hours, with less than 15 minutes of hands-on time, and is designed to maximize library yield through high reaction efficiency and minimal sample loss.

The TrueAmp Library Preparation Kit supports a broad range of DNA input amounts, from as low as 100 pg up to 1 µg. This flexibility enables reliable library preparation across low-input and high-input sample types.

No, the TrueAmp Library Preparation Kit is not compatible with bisulfite conversion or EM-seq workflows. The enzymatic steps in the TrueAmp Library Preparation workflow include DNA repair, which can alter or remove native methylation signatures.

For methylation-sensitive applications such as bisulfite or EM-seq, we recommend using mechanical shearing (e.g., sonication) followed by the Twist cfDNA Library Preparation Kit, which is better suited for preserving methylation information.

The TrueAmp Library Preparation Kit is not recommended for cfDNA workflows. Since cfDNA is already fragmented, the enzymatic fragmentation step is unnecessary. While it is possible to skip fragmentation and proceed with end repair and dA-tailing, the one-pot workflow may not deliver optimal conversion efficiency for these short fragments.

For best performance and highest library conversion from cfDNA, we recommend using the Twist cfDNA Library Preparation Kit, which is specifically optimized for cell-free DNA.

Yes, the TrueAmp Library Preparation Kit performs well with degraded and FFPE-derived DNA. The workflow includes DNA repair activity, which can help restore damaged bases commonly found in FFPE samples.

As a result, users often observe improved library yield and recovery compared to workflows without repair. Some optimization (e.g., fragmentation time, PCR cycles) may be required depending on sample quality.

是。The TrueAmp fragmentation chemistry is broadly compatible with a range of double-stranded DNA inputs, including genomic DNA (gDNA), plasmids, and PCR-derived amplicons. Under consistent reaction conditions, high-quality DNA substrates, regardless of origin, typically yield comparable fragment size distributions, indicating minimal sequence or topology-dependent bias.

During development, fragmentation performance was evaluated using DNA ladders spanning a wide range of fragment sizes. This study demonstrates that the enzymatic fragmentation preferentially processes larger DNA molecules while maintaining representation of shorter fragments, resulting in a controlled size distribution without significant loss of diversity across the input population.

Yes, the TrueAmp Library Preparation Kit is well suited for targeted enrichment workflows, including hybrid capture applications. It generates high library yields and promotes uniform molecule conversion, which supports efficient and consistent downstream target capture performance.

Yes, the TrueAmp Library Preparation Kit is compatible with liquid handling automation platforms such as Hamilton systems. The streamlined workflow, reduced number of steps, and minimal hands-on time make it well suited for automation. Minor optimization (e.g., mixing, bead handling, or liquid classes) may be required depending on the platform and configuration.

No. The enzymatic fragmentation chemistry is designed to produce consistent fragment sizes across the recommended input range, so fragmentation time and temperature do not need to be adjusted based on DNA mass.

Under the same conditions, samples with similar DNA quality ranging from 0.1 ng to 500 ng will generate comparable fragment size distributions. As demonstrated above, human genomic DNA inputs from 0.1 ng to 500 ng fragmented at 32°C for 22 minutes produced similar final library sizes (~450–480 bp), with PCR cycle number increasing as input mass decreased.

Input DNA quality directly affects fragment size. High-quality, intact DNA produces predictable insert sizes, while degraded or pre-sheared DNA (e.g., FFPE) typically results in shorter fragments under the same conditions. In these cases, reducing fragmentation time or temperature and applying size selection may help achieve the desired insert size.

Fragment size can be controlled by adjusting fragmentation reaction temperature and incubation time. Increasing either parameter generally results in shorter DNA fragments, while reducing them produces longer fragments.

Because fragmentation performance can vary depending on thermocycler calibration, DNA input quality, and storage buffer composition, we recommend performing a small design of experiments (DOE) with your specific samples to determine optimal conditions prior to scaling up.

For additional guidance and example data, please refer to the Twist TrueAmp Library Preparation Kit technical note on insert size optimization.

Yes, fragmented and end-prepped DNA can be safely stored at either 4 °C or -20 °C (e.g., overnight) prior to ligation without any appreciable impact on library preparation performance as compared to samples processed immediately. This has been validated across technical replicates (n=3), where samples were stored under these conditions before ligation and showed consistent results.

是。Vortexing the 10X Twist Fragmentation Enzyme Mix prior to use is important to ensure the solution is fully homogeneous. Failure to properly mix the enzyme solution may lead to variability in fragmentation performance and inconsistent results.

The TrueAmp Library Preparation Kit is designed for flexibility and is compatible with a wide range of 3′ T-overhang adapters. Note that adapter quality impacts overall library preparation efficiency. For optimal performance, we recommend using validated Twist adapter systems:

●        Twist Universal Adapter System (SKU: 101308)

○    Requires PCR amplification with indexed primers to complete library construction.

●        Twist UMI Adapter System (SKU: 105041)

○        Requires PCR amplification with indexed primers.

○        Enables incorporation of Unique Molecular Identifiers (UMIs) for improved error correction and duplicate handling.

●        Twist Full-Length UDI Adapters (SKU: 107376)

○        Do not require PCR for index incorporation, as full-length P5/P7 sequences and indices are already present.

○        Optional PCR may still be performed for library amplification if additional yield is needed.

All three systems are fully compatible with the TrueAmp workflow. Selection should be based on your application needs (e.g., UMI requirements, workflow, or PCR-free indexing preference).

Adapter input should be adjusted based on DNA input amount and fragment size to maintain sufficient adapter-to-insert molar excess for efficient ligation while minimizing adapter-dimer formation. Twist Universal Adapters are supplied at 10 µM and recommended conditions in the protocol are designed to maintain an adapter-to-insert molar ratio of approximately ≥150:1 for robust ligation efficiency across a broad range of inputs. Too little adapter may reduce library conversion and yield, while excessive adapter can increase adapter-dimer carryover, particularly in low-input samples.

If optimization is required, titrate adapter input while monitoring final library yield and adapter-dimer levels. The optimal condition is the lowest adapter amount that maintains strong library yield with minimal adapter dimer.

For ultra-low input samples (<1 ng), additional optimization may be required to balance ligation efficiency against adapter-dimer formation, as reduced insert mass increases the likelihood of adapter self-ligation products. Conversely, for higher DNA input amounts, increasing adapter concentration beyond the recommended range may further improve ligation efficiency in some workflows.

Yes, contaminants such as salts, EDTA, ethanol, detergents, and extraction reagent carryover can impact enzymatic fragmentation. These may either inhibit or enhance enzyme activity, leading to inconsistent or shifted fragment sizes.

In some cases, fragmentation can be tuned by adjusting incubation time or temperature to compensate. However, if inhibitors significantly disrupt enzyme activity and no library is generated, we recommend performing an additional DNA cleanup before library preparation.

Three independent manufacturing lots were evaluated to assess fragmentation variability, with an additional operator performing replicate testing on a separate day to characterize operator-to-operator effects.

Fragmentation with the TrueAmp Library Preparation Kit demonstrates high reproducibility within a given lot, with technical replicates (n=8) showing tight fragment size distributions and a standard deviation of ~7–10 bp (~2% CV). Across manufacturing lots, modest shifts in the mean fragment size are observed (~30–40 bp, or ~5–8% deviation), while maintaining consistent distribution shape and overall library performance. Operator-to-operator variability can introduce similar shifts in the average fragment size, as observed within the same lot; however, these differences should not meaningfully impact the distribution profile or downstream performance. 

是。The TrueAmp reagents have been tested through up to four freeze-thaw cycles with no observed impact on performance. These studies were conducted over multiple days to reflect typical laboratory use. As a best practice, repeated freeze–thaw cycles should still be minimized when possible.

The TrueAmp Library Preparation Kit has a shelf life of 12 months when stored as recommended. This is supported by accelerated aging studies conducted over 13 months, which showed no measurable impact on performance or stability.

Yes, we offer flexible customization options to support a wide range of specialized requirements or unique applications. For more information or to discuss your specific requirements, please contact our team.

The TrueAmp Library Preparation Kit is not recommended for workflows where DNA is already sheared or where native DNA modifications must be preserved. This includes applications such as cfDNA library preparation and methylation-sensitive workflows (e.g., bisulfite or EM-seq). For these use cases, alternative workflows or kits optimized for those applications are recommended.

The insert is the DNA of interest generated during fragmentation. The insert size refers to the length of this DNA region. The fragment (or sequencing fragment/library) includes the insert plus the sequencing adapters ligated to both ends. Therefore, fragment size = insert size + adapter sequences. Traditional adapter sequences are roughly 65-70nt long but dependent on the presence of UMIs or length of indexes. In paired-end sequencing, reads are generated from both ends of the insert. If the insert is longer than the combined read length, a portion in the middle remains unsequenced. If the insert is shorter, reads may overlap or extend into adapter sequences.

Insert size impacts library yield, sequencing performance, and downstream applications. Smaller fragments typically amplify more efficiently and can result in higher yields, while larger fragments may reduce cluster generation efficiency on some sequencing platforms. Insert size also influences data quality metrics such as coverage uniformity, duplication rates, and alignment efficiency.

This is especially true in targeted enrichment workflows, including multiplexed captures, where the same mass input (e.g., 500 ng) represents a different number of molecules depending on fragment size.  Smaller libraries contain more molecules than larger ones at the same mass, which can affect capture efficiency and sample balance.

Additionally, larger insert sizes in target enrichment workflows can increase the proportion of near-bait reads, as longer fragments extend beyond the probe-binding region. Insert size should also be considered relative to sequencing read length. For paired-end sequencing (e.g., 2×75 bp or 2×150 bp), inserts that are too short may result in overlapping reads or sequencing into adapter regions, reducing usable data. Selecting an appropriate and consistent insert size for your application helps ensure optimal sequencing performance and reliable downstream analysis.

Shorter-than-expected fragments typically result from degraded or pre-sheared input DNA, excessive fragmentation time or temperature, or buffer components (e.g., salts) that increase enzymatic activity. We recommend reducing fragmentation time or temperature and confirming input DNA quality to help achieve the desired insert size. If needed, performing a cleanup step prior to library preparation may improve consistency.

If your DNA has been sufficiently fragmented, the presence of multiple peaks, especially those appearing larger than the expected fragment size, is typically an artifact of library preparation rather than true size distribution:

1. PCR overamplification and heteroduplex formation.
During excessive PCR cycling, DNA strands from different templates can anneal to each other, forming heteroduplexes. These structures migrate more slowly during capillary electrophoresis (e.g., TapeStation or Bioanalyzer), which can create the appearance of larger fragment sizes or additional peaks.

For additional information, please refer to the technical note on heteroduplexes: https://www.twistbioscience.com/resources/white-paper/heteroduplexes-affect-library-size-determination-without-impacting-targeted

Overcycling can introduce heteroduplex artifacts in adapter-ligated libraries. Samples of the gDNA library were subjected to 2x increments in rounds of PCR amplification. Top/Middle: Schematic of homoduplex and heteroduplex formation during PCR amplification. With appropriate amplification, library molecules form homoduplex products. With excessive PCR cycling, related but non-identical library molecules can misanneal, forming heteroduplex species that migrate aberrantly during electrophoresis. Bottom: Bioanalyzer electropherograms representing increasing PCR cycles (left to right, 4 - 12 cycles). PCR libraries show progressively broad, irregular peaks and apparent high-molecular-weight species, consistent with heteroduplex formation from overamplification.

2. Adapter structure effects (full-length Y adapters)
Libraries constructed with full-length Y adapters (containing both P5 and P7 sequences) can exhibit altered migration behavior due to their partially single-stranded structure. This can cause fragments to appear larger than their true size during electrophoretic analysis.

These 2 effects can be validated by performing a short “reconditioning PCR”: Use a small number of cycles (e.g., 2–5 cycles) with the inclusion of a high concentration of primers
This step promotes the formation of fully matched homoduplexes, which migrate more accurately during electrophoresis. After this treatment, the apparent fragment size distribution should shift closer to the expected profile.

Resolution of library fragment size following PCR amplification. 250ng of gDNA was fragmented at 25°C for 15 mins for a PCR-free protocol (A) TapeStation trace of a library immediately following ligation of Twist Full-Length UDI Adapters. The electropherogram shows multiple broad and irregular peaks, reflecting heterogeneous migration behavior caused by the Y-shaped adapter structure. These structural features can result in apparent high-molecular-weight species and poor size resolution. (B) TapeStation trace of the same library after 3 cycles of PCR amplification. PCR converts adapter-ligated molecules into fully double-stranded homoduplex products, resolving structural heterogeneity and yielding a defined fragment size distribution.

Low library yield can result from low input DNA, poor DNA quality, over- or under-fragmentation, or the presence of inhibitors that affect enzymatic steps. Additional factors such as improper size selection, incomplete ethanol removal, over-dried SPRI beads, or insufficient mixing during reactions may also reduce yield. Ensuring clean DNA input, optimizing fragmentation conditions, and following recommended SPRI handling and mixing practices should improve performance.

Adapter dimers or short fragments are typically observed with low input DNA, excess adapter concentration, or insufficient size selection. Additional causes include improper adapter handling (e.g., degradation or incorrect dilution) or pre-mixing adapters with ligation reagents. Optimizing adapter concentration, ensuring proper adapter handling, and performing appropriate SPRI cleanup (e.g., adjusting bead ratios or repeating cleanup) can help reduce these artifacts.

As a general starting point, the baseline fragmentation times and temperatures in the protocol are recommended to achieve insert sizes in the 200-250 bp range. Fragmentation behavior can vary depending on thermocycler calibration, DNA quality, and buffer composition. For best results, we recommend performing a small time-course optimization (DOE) using your specific samples and follow this decision tree for optimization of the target insert size.

The number of PCR cycles depends on input DNA amount and desired library yield. Lower input samples typically require more cycles to generate sufficient material, while higher input samples require fewer cycles to minimize duplication and preserve library complexity. Optimization may be required to balance yield and sequencing performance.

热启动适配体通过在低温下形成二级结构（如室温或在实验台上进行配制时）来工作。这种二级结构与聚合酶结合，使酶活性失活。在更高温度下（如在 PCR 热循环期间），适配体的二级结构变性，从而释放对酶的束缚。

作为质量控制 (QC) 流程的一部分，我们设置了在 37℃ 下长达 10 小时的孵育步骤，以确保低温配制操作不会影响其性能。

全新设计的 Twist TrueAmp 聚合酶预混液经过工程化改造和优化，具有高产出和稳健的性能，可高效扩增起始量低至 100 fg 的 DNA 及标准起始量样本，为不同类型的样本提供单一、可靠的解决方案。

The Twist TrueAmp Polymerase is engineered for enhanced proofreading, resulting in an ultra-low error rate and significant reduction in sequencing artifacts such as C>T substitutions. Internal testing has shown an absolute error rate of 1.17 × 10⁻⁶. Please take a look at our product sheet for more information.

我们已验证了其在长读长测序上的性能与当前市场领先的聚合酶相当，可直接替代使用。但目前，我们的研发重点已转向专注于提供超高保真度的长读长解决方案。

Twist TrueAmp 聚合酶预混液仅供研究使用。

Twist TrueAmp 聚合酶预混液在 -20℃ 条件下运输和储存。

Twist TrueAmp 聚合酶预混液可耐受高达 20 次冻融循环，且性能无变化。该预混液有效期为 2 年。

是，工程化连接酶无需依赖聚乙二醇（PEG）等拥挤剂即可实现最大连接效率。PEG is automation-unfriendly, increases variability/viscosity, and negatively impacts downstream bead-based purification (SPRI). 我们建议使用 0.9 倍 SPRI 作为 cfDNA 回收的优化比例（~150-167 bp）。For shorter cfDNA fragments, we recommend increasing the SPRI ratio to 1.0X or 1.1X.

是的，该试剂盒的推荐方案允许使用 35 μl 的 cfDNA 输入量。已对高达 50 μl 的大量样本进行测试，未发现对试剂盒性能产生负面影响。在此情况下，我们建议减少连接反应中的水量，以补偿末端修复过程中增加的量。

不，该试剂盒可处理从 0.5 ng 到高达 500 ng 的广泛样本输入范围。

接头总量和 PCR 循环数需根据样本输入量进行调整。

是。The kit can be used with low input DNA samples that do not need fragmentation, or with pre-fragmented input DNA such as samples derived from FFPE material.

In tests performed with moderate DIN (3-5) FFPE curls from 4 sample sources (HG002, HG003, HG004, and Structural Multiplex Standard),  results show that the cfDNA library preparation kit can be used on FFPE material and adapted for solid tumor workflows using cfDNA Library Prep Kit and the standard hybridization v2 protocol.

Note that DNA fragmentation is still necessary even for samples with a DIN score <2. Additionally, DNA input ranges will need to be increased to accommodate the availability of DNA from FFPE extraction (~100-500 ng per 15 μm curl).

我们建议使用凝胶电泳图谱分析法（例如 Bioanalyzer 高灵敏度 DNA 试剂盒（Agilent））来分析输入 cfDNA 的大小分布，以确保其不含可能从 cfDNA 提取过程中遗留的高分子量（HMW）DNA。HMW DNA will negatively impact the accurate quantification of cfDNA input amounts. We also recommend quantifying input cfDNA with a Qubit high-sensitivity dsDNA assay (Thermofisher) for accurate DNA quantification. 

是，该试剂盒确实可以使用 UMI 来制备 cfDNA 文库。

我们发现，靶向富集中的文库输入质量会影响深度测序文库的复杂性，而这一倍数是基于实验数据确定的。这种方法确保了富集过程的最大效率。

如需更多详情，请参阅我们的 cfDNA 文库制备试剂盒数据手册，点击此处查看： https://www.twistbioscience.com/resources/technical-document/cfdna-library-prep-kit-datasheet

我们建议参考我们的数据手册以确定最佳 cfDNA 质量输入量： https://www.twistbioscience.com/resources/technical-document/cfdna-library-prep-kit-datasheet

最佳的 cfDNA 输入量应通过综合考虑目标 VAF 下的所需灵敏度、双重检测要求、组合内容以及可用的测序资源等因素来确定。

用于靶向富集的文库质量是根据输入到文库制备中的 cfDNA 样本质量计算得出的。在进行单重或 8 重杂交时，使用每个扩增的标签文库的浓度来计算杂交所需的每个文库的体积（单位为 μl）。此信息也可在该方案的附录 B 中找到：https://www.twistbioscience.com/resources/protocol/twist-cfdna-target-enrichment-standard-hybridization-protocol

推荐的最大靶向富集文库产量为 12.8 μg。When pooling libraries from variable starting mass inputs, each library should have the same input mass added to the pool for uniform sequencing allocation post-capture.

要计算该质量，将最高质量输入乘以 80。例如：

●       With an 8-plex where the highest sample mass input is 20 ng, pool 1,600 ng from each library for a total of 12.8 μg.

●       With an 8-plex where the highest sample mass input is 10 ng, pool 800 ng from each library for a total of 6.4 μg.

cfDNA 文库制备试剂盒的工作流程可在不到 3 小时内构建文库。

建议的 DNA 输入为 1 - 20 ng cfDNA，悬浮于分子生物学级用水、10 mM Tris-HCl pH 8.0 或缓冲液 EB 中。

针对靶向富集的最小质量输入推荐值仅基于单重样本的 cfDNA 文库制备过程中的质量输入（见公式）。这是推荐的最低通过标准。将靶向富集质量增加至 12.8 μg 是可接受的。

cfDNA 文库制备试剂盒采用干冰运输，收到后需在 -25°C 至 -15°C 的温度范围内储存。

该试剂盒可制备 cfDNA 文库，既可使用通用接头进行 PCR 反应（搭配 UDI 引物），也可使用无 PCR 和 WGS 应用的全长 UDI 接头（详见此处的 cfDNA 全长 UDI 方案：

https://www.twistbioscience.com/resources/protocol/twist-cfdna-library-preparation-kit-twist-full-length-udi-adapters).

如需更多信息，请参阅 https://www.twistbioscience.com/products/ngs/library-preparation/universal-adapter-system。

此外，该试剂盒的分子生物学组分应与其他供应商提供的接头和引物兼容。However, the adapter must contain a 3’ T overhang to facilitate the A/T ligation with the 3’ A overhang in the library molecules. 

最终文库产量在不使用 Twist 优化解决方案时可能会略微降低。

我们建议降低浓度以防止超过 12.8 μg 的限值。

按照推荐的储存和处理方式，在所注明的保质期之前该产品将保持其全部性能。The product has been tested with up to eight freeze/thaw cycles, and shown no impact to expected performance.

cfDNA 文库制备试剂盒构建的标签文库可用于全基因组测序（WGS）实验，或与 Twist 靶向富集试剂盒一起用于下游实验，并在 Illumina 下一代测序（NGS）系统上进行测序。

该 cfDNA 靶向富集试剂盒支持 1 重（单重）或 8 重杂交捕获。

我们观察到基于目标捕获指标的性能下降。这种性能下降与脱靶效应的增加相关。

如果使用过多的 PCR 循环，可能会导致过度扩增。Once PCR primers are depleted, library fragments may be single stranded or form heteroduplexes that appear as high molecular weight fragments in capillary gel electrophoresis instruments. 这些峰值与早期 PCR 循环中预期出现的 cfDNA 二核小体峰值不同。We recommend starting with the number of PCR cycles recommended in the protocol (https://www.twistbioscience.com/resources/protocol/twist-cfdna-library-preparation-twist-umi-adapter-system) and reducing PCR cycles if overamplification is observed.

该试剂盒提供所有必要的试剂，可制备多达 960 个可用于测序的文库；然而，客户需要准备常用的实验室设备和试剂，如移液器吸头、磁力支架和 EDTA。完整列表参见方案。

另外，客户负责提供样本拆分计算资源。Twist 会提供样本拆分指南。

由于标签跳跃机制与接头交换相关联，因此 96-Plex 文库不太可能受到影响，因为它们使用内联 (inline) 条形码。此外，文库制备过程中，根据粒子的最终尺寸选择步骤，应尽量减少最终文库中出现游离接头。

目前没有。

是的，但解复用必须独立完成，每个文库类型进行一次。具体说明见解复用指南。

此时，无法购买单个试剂盒和组件。

使用您所使用的测序平台的默认推荐上样浓度。应按 Agilent 生物分析仪中主要目标峰的范围分析，测定摩尔浓度。根据定量结果（而非模式峰大小或默认摩尔浓度估计值）进行稀释。

可以在 Twist 的电子商务网站上找到 Twist 96-Plex 文库制备试剂盒。在 SARS 应用程序中，搜索 104951 或 104950；会出现 Twist 96-Plex 文库制备试剂盒，4 x 96 个样本 ($4,300) 或 10 x 96 个样本 ($9,600)。

试剂盒包含用于“A”反应的两个引物板。高 GC 孔板具有随机引物，可针对 GC 含量 > 50% 的序列调整，而低 GC 孔板的引物可针对 GC 含量 < 50% 的序列调整。Users may optimize their workflow using either of these plates, or combining them in a 1:1 ratio for 40-60% GC. 

试剂盒的配置为每批处理多达 96 个样本。“A”板中的空孔无法保留用于后续批次，因此孔板填充不完全会降低您的成本效益。

如果您想要一次处理少于 96 个样本，我们建议用重复样本填充孔板的剩余孔，这样可提高测序结果的可靠性。

是。在这种情况下，不应读取识别孔板的 6 核苷酸 Illumina 小 RNA TruSeq i7 标签。要么不执行标签读取，要么使用“--use-bases-mask”选项忽略 6nt 标签读取。这将导致整个测序运行产生一组 FASTQ。更多信息，请参见解复用指南。

我们提供两种试剂盒规格配置。

第一种规格可处理多达 960 个样本；试剂盒包含足够用于 10 个 96 孔板的反应物。试剂盒不需要一次性全部使用，但建议每次测序运行至少使用一个孔板。

最新推出的配置为 4 x 96 配置试剂盒，可以完成 384 个样本。该试剂盒与 10 x 96 试剂盒的工作流程相同，且每个样本的成本效益同样可观，但整体价格和样本量均更低，更适合希望试用本产品的新客户，以及处理零散项目（少于 960 个样本）的客户。

您可以在 Twist 网站上获取更多的产品信息（包括产品列表、方案和解复用指南的链接）。

推荐的输入量为 50 ng，应产生约 200 ng 的最终文库。若使用的粒子尺寸选择条件不同，输出会因而改变。建议输入高分子量 DNA。我们建议避免在此试剂盒中使用降解样本，如 FFPE。

解复用 Twist 96-Plex 文库与标准过程不同。单个样本条形码作为 R1 的一部分排列，而不是在标准 i5 位置。正因为如此，Illumina 的 bcl2fastq2 和 BCLconvert 工具不能识别单个样本条形码，在解复用过程中，其根据单个 i7 标签，将所有孔组合在一个单一的孔板级 FASTQ 文件中。

为了解决这个问题，96-Plex 使用者必须按照两步解复用过程进行操作。首先，每个循环的 BCL 文件必须转换为孔板级 FASTQ，然后将孔板级 FASTQ 文件第二次转换为样本特定的 FASTQ 文件。我们建议使用 fgbio DemuxFastqs 开源软件，生成每个样本的 FASTQ。更多信息请参见 Twist 网站上的解复用指南。

96-Plex 试剂盒自生产之日起有效期为一年。

可在 Twist 网站上获得 96-Plex 文库制备方案。

试剂盒与 Illumina 平台兼容。用全长 Illumina 接头制备双链文库，用于单端或配对末端测序。此时，最终文库与其他测序技术不兼容使用。

TruSeq 接头序列应用于 Illumina bcl2fastq2/bclconvert 孔板级解复用过程中的接头修剪。使用 fgbio 解复用工具，您可以在样本级解复用过程中修剪 R1 上的内联 (inline) 条形码与读段上的合成随机序列。

试剂盒中包括 CpG 甲基化 pUC19 DNA 和未甲基化 Lambda DNA。这些质控品可用于测定文库制备过程中酶转化的效率。

注：内部质控品不应包含在捕获工作流中，除非特定质控品的探针被添加到富集组合中。

是，多重测序最多支持 8 重。我们建议在单重捕获中使用 200 ng 的文库， 8 重捕获则使用 1500 ng（或每个 187.5 ng）。

否。杂交捕获必须采用 Twist Fast 杂交系统进行。

使用该试剂时，不会对任何杂交选择指标造成损害。它以一种可变的方式减少脱靶量，具体决于定制甲基化组合的目标区域和输入基因组 DNA 的甲基化状态。在某些定制组合，脱靶率可能会减少 50%。

如表所示：

预期最终文库产量在 50-75 ng/ul 之间。产量将取决于初始投入的质量。

350 到 450 bp

我们建议使用最低 10-20 ng 的高质量 DNA，最高为 200 ng。

200 到 300 bp

当构建文库以进行甲基化转化时，Twist 仅支持该应用的机械片段化。

可进行如下改进，以优化杂交捕获：

• 杂交时间 - 建议从 2 小时开始，在 30 分钟至 4 小时之间改进。
• Fast Wash Buffer 1 Temperature – Start with 65°C, adjust between 63–66°C.

Yes; however, increases in library complexity may not be as dramatic as those seen with low input cfDNA.

是的，在文库制备步骤中，Twist 甲基化 UMI 可作为替代品使用。

是的，Twist 技术支持部门可根据购买后的要求提供 UMI 序列。给 customersupport@twistbioscience.com 发邮件寻求支持。

Yes, bioinformatic processing steps are outlined in the product sheet.

5 个可操作碱基和 2 个跳过碱基。

Twist 甲基化 UMI 接头由 32 对不连续的适配体组成，具有直列 UMI 序列，即在测序引物结合位点之后出现。

Twist 通用封闭剂与来自 Illumina® 的 TruSeq® 样式文库试剂盒相容，采用单标记和双标记方案，并且有各种条码和 UMI 长度。要了解更多信息，请下载我们的通用封闭剂产品说明书。

我们建议杂交 DNA 的最大输入量为 4 ug。添加更多的 DNA 可能会降低封闭剂的效率，从而可能会导致脱靶读数较高。

NGS 试剂盒中捕获后扩增使用的引物序列为：

P5 引物：AATGATACGGCGACCACCGA

P7 引物：CAAGCAGAAGACGGCATACGA

我们引物的浓度为每个引物 10 uM。捕获后的 PCR 中最终引物浓度为 0.5 uM。