User Manual. NGS Library qpcr Quantification Kit (Illumina compatible)

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1 NGS Library qpcr Quantification Kit (Illumina compatible) User Manual 384 Oyster Point Blvd, Suite 15 South San Francisco, CA Phone: 1 (888) MCLAB-88 Fax: 1 (650)

2 Contents Description List of Components Additional Materials Required qpcr NGS Library Quantification Procedure Best Practice

Description: Accurate quantification of the molarity of the DNA library is required for next generation sequencing (NGS) to ensure high quality reads and efficient data generation.

3 Description: Accurate quantification of the molarity of the DNA library is required for next generation sequencing (NGS) to ensure high quality reads and efficient data generation. Polymerase Chain Reaction (PCR) is a highly sensitive approach for quantifying a DNA NGS library and uses a minimal amount of material compared to other quantification approaches. It tracks target concentration as a function of PCR cycle number in order to derive a quantitative estimate of the initial template concentration in a sample. MCLAB s qpcr NGS Library Quantification Kit (Illumina - compatible) provides a fast and reliable method for determining the concentration of DNA library using real-time PCR (qpcr). The kit is compatible with libraries designed for the Illumina system. The kit includes 2X HotSybr PCR Reaction Mix which contains a mutant Taq DNA polymerase, dntps, and the double-stranded DNA-binding dye SYBR Green I for detection. The kit also includes primers and a defined, pure, linear dsdna standard of known concentration that is used to generate a standard curve to which the library samples are compared. Ideally, the control template should be as similar as possible to the libraries for quantification, in consideration of the template size, GC content and library type (e.g., genomic, ChIP-Seq, etc.). Unfortunately the GC content of a given library is not always known. It is recommended to generate a titration flowcell for the test library (especially for first time libraries) based on qpcr quantification results. An overview of the quantification protocol is shown in Figure 1. To adapt this protocol when using different qpcr platforms, an optional passive reference dye (ROX) is provided in a separate mixture, such as Regular level of ROX for qpcr Machines of ABI 7000, 7300, 7700, 7900; Low level of ROX for qpcr Machines of ABI 7500, Stratagene Mx 3000P, Mx 3005P and ROX Free for qpcr Machines of BioRad icycler, MiniOpticon, Opticon 2, Chromo4, iq5; Roche LightCycler 480; MJ Research DNA Engine Opticon 2, Chromo4; Corbett Roto-gene 3000, List of Components: Sufficient reagents are supplied in MCLAB s NGS Library qpcr Quantification Kit (Illumina -compatible) for 400 reactions to quantify NGS libraries for high-throughput sequencing through the Illumina platform. Upon receipt of the kit, immediately store the components at 20 C in a freezer without a defrost cycle. Once the 2 HotSybr PCR Reaction Mix has been thawed, it can be stored at 4 C for up to one month or returned to - 20 C for long term storage. The PCR Mix is light sensitive and protect the solution from light whenever possible. When stored and handled correctly, all components will be stable for at least six months from the date of receipt. NGS Library qpcr Quantification Kit (400 reactions) 2X HotSybr PCR Reaction Mix, 4x1.25 ml (HSM400 or HSM400LR or HSM400RF) Primer Mix, 200 μl DNA Library Standard, 80 μl Figure 2 Components of MCLAB qpcr NGS Library Quantification Kit Figure 1 Overview of the library quantification Workflow 2

Additional Materials Required: The following equipment, consumables and materials are required, but not included: Real-time PCR instrument Vortexer Benchtop cooler (optional) Benchtop centrifuge

4 Additional Materials Required: The following equipment, consumables and materials are required, but not included: Real-time PCR instrument Vortexer Benchtop cooler (optional) Benchtop centrifuge Pipettors, positive displacement or air-displacement PCR plate (or tubes) and adhesive seals Pipette tips, nuclease-free Nuclease-free PCR water DNA Storage Buffer (Cat# DSB-100, DSB-200) qpcr NGS Library Quantification Procedure: Experiment preparation - Prepare at least five serial (10-fold) fresh dilutions of the DNA standard with each qpcr run (triplicate each reaction). - Include two 2-fold serial dilutions of the DNA library (in triplicate), with the molarity of the more concentrated dilution estimated to be between 1 and 10 pm. The rough concentration of the library can be determined by running a sample on a gel or from the OD 260 reading on a spectrophotometer, or it can be estimated based on data gathered during library preparation. Prepare qpcr Reaction Mix 1. Set up qpcr reaction Mix on ice for each reaction as below: Component 2x HotSybr* Primer Mix PCR-grade Water Total Volume Volume 12.5 μl 0.5 μl 9 μl 22 μl * Make sure you have appropriate qpcr mix that is compatible with your qpcr instrument. For multiple reactions, including DNA Standard, library samples and non-template control, master mix should be made with at least 5% extra reagents. 2. Using a multichannel pipette, and accurately transfer 22 μl reaction mixture into each well. 3. Add 3 μl of the standard dilutions, the unknown library dilutions, or water to corresponding wells. 4. Seal the plate using an appropriate adhesive seal. Place the plate on the plate adapter and centrifuge at 250 xg for 1 minute. 5. If you are using 96-well plate, please refer to Figure 3 for one of assay design formats. It is recommended to set up triplicate qpcr reaction for each sample. Prepare 10-fold Serial Dilutions of DNA Standard 1. Add 5 μl of DNA standard (Standard 1) into 45 μl of DNA Storage Buffer (not included) to make a 10-fold dilution for Standard Vortex the dilution to mix the sample and spin down. 3. Transfer 5 μl Standard 2 to 45 μl of DNA Storage Buffer to make a further dilution (Standard 3). 4. Repeat steps to prepare Standard 4, 5 and 6. The diluted samples can be stored at 4 C for up to a day. Prepare Serial Dilutions of the DNA Library It is recommended to make fresh dilutions of the unknown library template for each qpcr run. 1. Based on concentration information from library construction, dilute libraries to approximately 2 nm in DNA Storage Buffer for quantification. 2. Transfer 2 μl of 2 nm unknown library template to 998 μl of DNA Storage Buffer to make a 500 fold dilution for an approximate concentration of 4 pm. 3. Vortex the dilution to mix the sample and spin down. 4. Transfer 10 μl diluted library to 10 μl of DNA Storage Buffer to make a second 2-fold dilution. Figure 3 Example of qpcr assay design layout using 96-well plate Run the qpcr Protocol 1. Place the plate in the thermal cycler. 2. Select thermal profile provided by the software for absolute quantification assay with SYBR as reported. Typical qpcr cycling conditions are as follows: One cycle at 95 C for 10 minutes; Repeat 35 cycles of 95 C for 15 seconds and 60 C for 45 seconds (signal acquisition), 72 C for 15 seconds (optional); Followed by the melting curve program (optional); 4 C hold. 3. Start the run. Calculate Library Concentration 1. Check result of non-template control reaction. There should be no amplification in these wells. 1-(650)

5 2. Check result of the standards. There should be good amplification. The efficiency of amplification of the standards is between 90% and 110% (a slope of 3.6 to 3.1) and the R2 is over Generate a standard curve from the standards by plotting the quantification cycle (Cq) values against the log initial concentration (3 μl Standard 1 at 20 pm) with installed software. 4. Check the results of the unknown library templates and make sure there are good amplifications (i.e. the efficiency of amplification is between 90% and 110%, with a slope of 3.6 to 3.1) and the R2 is over Use the average of the triplicate data points corresponding to the most concentrated library DNA dilution that falls within the dynamic range of the DNA Standards to calculate the concentration of the undiluted library. If one of the three replicates appears to be an outlier, it may be omitted from the calculation. If more than one of the three replicates are outliers, the sample dilution and assay should be repeated. 6. Calculate the concentration (X) of diluted unknown library templates based on the standard curve generated from the standards. 7. Perform a size adjustment calculation (Y) to account for the difference in size between the average fragment length of the library (determined by Bioanalyzer or other equivalent methods) and the control (523 bp). Y=523 * X/(average length) 8. Calculate the initial concentration (Z) of the undiluted library (~2 nm) by taking into account of the relevant dilution factor (500x for ~4 pm dilution and 1000x for second dilution). Z= (dilution factor) * Y 9. Use the calculated initial concentration (Z) of the undiluted library to prepare expected dilution for flow cell loading and cluster generation. The DNA Storage Buffer is suitable for small amounts of next generation sequencing (NGS) library long-term storage, which would decrease the cluster numbers over time. It is also appropriate for making highly dilute NGS libraries during quantification assay. It is recommended to handle MCLAB's DNA Storage Buffer with low adhesion/binding pipette tips and tubes for better performance. It is optional to perform serial dilutions for unknown library samples. One of the serial dilutions must fall within the dynamic range of the standards. The dilution series also provides information about technical accuracy and/or qpcr efficiency. It is acceptable to remove outliers from a replicate group if differences are over 0.5 quantification cycle (Cq). Dissociation (melting) curve analysis is also optional. It may provide a useful indication of possible primer and/or adaptor dimer contamination of libraries. Best Practice When preparing libraries for sequencing, it is important to follow good molecular biology practices, especially taking extra care to avoid DNA cross-contamination and avoid pipetting errors. Make sure the reaction solutions containing the reference dye are protected from light. Do not use pure water to prepare dilutions of the DNA standard and unknown libraries. It is highly recommended to use DNA storage Buffer (not included) for serial dilutions. It contains proprietary stabilizers and ingredients preserving high quality of DNA samples and preventing absorption of the target DNA to plastic tubes upon repeated freeze-thaw cycles. 4

Absolute Mouse Telomere Length Quantification qpcr Assay Kit (AMTLQ) Catalog #M reactions

Absolute Mouse Telomere Length Quantification qpcr Assay Kit (AMTLQ) Catalog #M8918 100 reactions Product Description Telomeres are repetitive nucleotide elements at the ends of chromosomes that protect