μCaler - Nanodigmbio (Nanjing) Biotechnology Co., Ltd.

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Title: How to detect 0.005% mutation ? A：① Increase the sample input
The lower limit of mutation detection corresponding to different experimental conditions

Library conversion rate	Sample Input
Library conversion rate	1 ng	10 ng	20 ng	30 ng	50 ng	100 ng	200 ng	300 ng	500 ng	1000 ng
20%	5.000%	0.500%	0.250%	0.168%	0.100%	0.050%	0.025%	0.017%	0.010%	0.005%
30%	3.400%	0.340%	0.170%	0.113%	0.068%	0.034%	0.017%	0.011%	0.007%	0.003%
50%	2.000%	0.200%	0.100%	0.067%	0.040%	0.020%	0.010%	0.007%	0.004%	0.002%
100%	1.000%	0.100%	0.050%	0.033%	0.020%	0.010%	0.005%	0.003%	0.002%	0.001%

When the library conversion rate is 20%, 0.005% of the mutations can be detected with a sample input of 1000 ng;
When the library conversion rate is 50%, 0.005% of the mutations can be detected with a sample input of 400 ng;
②Increase the number of mutation sites

How to improve the detection rate of low-frequency mutational signals with NGS protocols?

Title: How to improve the detection rate of low-frequency mutational signals with NGS protocols?

① Increase the sample input:The more sample input , the copies of support low-frequency mutation detection will be more；

② Increase the number of tracked mutations: the higher the number of mutation sites, the probability of detection will be higher.

Note：a. The more sample input, the copy number that supports low-frequency mutation detection will be more higher b. For the same sample size, the higher the number of mutation sites, the higher the detection probability;

Abbosh C, Birkbak N J, Swanton C. Early stage NSCLC—challenges to implementing ctDNA-based screening and MRD detection[J]. Nature Reviews Clinical Oncology, 2018, 15(9): 577-586.
Van Der Pol Y, Mouliere F. Toward the early detection of cancer by decoding the epigenetic and environmental fingerprints of cell-free DNA[J]. Cancer cell, 2019, 36(4): 350-368.

The hybridization time can be shortened?

Title: The hybridization time can be shortened? 30 min-16 hr hybridization capture performance is basically same, it is recommended to choose 1 hr .

Does the production of μCaler MRD Solution depend on imports, and is there a risk of supply ?

Title: Does the production of μCaler MRD Solution depend on imports, and is there a risk of supply ? The core components of μCaler MRD Solution are all produced domestically , so there is no need to worry about the supply.

The pre-libraries prepared by NadPreps kits or prepared by third-party kits can be used for hybridization capture and subsequent operations according to the protocol of μCaler MRD Solution (for Illumina ®)?

Title: The pre-libraries prepared by NadPreps kits or prepared by third-party kits can be used for hybridization capture and subsequent operations according to the protocol of μCaler MRD Solution (for Illumina ®)? The total solution from μCaler series is recommended.

Is hybrid capture possible with pre-libraries below 500 ng?

Title: Is hybrid capture possible with pre-libraries below 500 ng? Yes

How does μCaler MRD Solution achieve 3-day fast delivery ?

Title: How does μCaler MRD Solution achieve 3-day fast delivery ? Relying on the precise control of the whole process of own synthesis factory, A: Relying on the precise control of the whole process of its own nucleic acid synthesis factory.Start the high-throughput automatic synthesis of Probe from the first day of receiving the MRD order, and complete the modification synthesis, aminolysis, elution quantification, quality control (if the quality control standard is not met, the coincidence will be started simultaneously), and sub-packaging on the second day. and so on; on the third day, the overlapping and sub-packaging, draining, sub-packaging, Panel production and product packaging and delivery, etc. will be completed in sequence; ensure product can be delivery within 3 working days.

What are the possible reasons for relatively high library yield after hybrid capture with μCaler? How to solve it?

Title: What are the possible reasons for relatively high library yield after hybrid capture with μCaler? How to solve it?

Possible Reason	Solution
Relatively high starting input	Quantification by Qubit™ 3.0 is recommended.
Relatively high amplification cycles	Explore the optimal amplification cycles according to the recommended protocol in the operating guideline.
The elution is not performed according to operational guideline, leading to serious off-target.	The elution is performed in strict accordance with the requirements of operating guideline.

What are the possible reasons for Low library yield after hybrid capture with μCaler? How to solve it?

Title: What are the possible reasons for Low library yield after hybrid capture with μCaler? How to solve it?

Possible Reason	Solution
Inaccurate starting input	In case of lower actual starting input, precise quantification by qPCR is recommended.
Reaction mixture is not completely transferred out when pipetting.	Transfer all reaction mixture from each step to a new tube when pipetting.
Some magnetic beads are discarded during hybridization and bead purification	Avoid too intense operation during the elution, and discard supernatant without losing any magnetic beads.

What are the possible reasons for Low on-target rate after hybrid capture with μCaler? How to solve it?

Title: What are the possible reasons for Low on-target rate after hybrid capture with μCaler? How to solve it?

Possible Reason	Solution
A lot of air bubbles are generated during elution	When using a pipette, it is necessary to gently pipette up and down to mix well to prevent the air from entering; If air bubbles are generated, first use the pipette to remove the air bubbles above the solution and then discard the supernatant.
PCR tube cap is not replaced	Replace the PCR tube and cap in strict accordance with the instruction for use.
Reagent is not fully mixed	Mix the reagents thoroughly before use according to the operation guideline and allow to warm up at the corresponding temperature.