μCaler - Nanodigmbio | Simplified Precision in NGS

What are the possible reasons for low pre-library yield? How to solve it?

Possible Reason	Solution
Input amount of nucleic acid is too low	If the sample is a co-extracted total nucleic acid, quantiﬁcation of RNA and DNA should be performed separately using Qubit, and library ampliﬁcation should be carried out according to the recommended cycles in the manual.
	During the experiment, RNase-free consumables must be used, and the operating environment should be free of RNase contamination, as this can aﬀect the eﬃciency of RNA reverse transcription.
	If the nucleic acid sample contains excessive amounts of chelating agents, guanidine salts, phenol, proteins, ethanol, or other impurities, it can impact the activity of RNA reverse transcriptase and the eﬃciency of DNA enzymatic digestion. It is recommended refer to Step 7: Post-ampliﬁcation Clean-up to purify the nucleic acid samples using 2X NadPrep SP Beads, replace the elution reagent with Nuclease Free Water, and then proceed with library preparation.

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?

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?

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 ?

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 ®)?

The total solution from μCaler series is recommended.

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

Yes

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?

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 captured-library yeild? 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.