| Possible Reason | Solution |
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The ratio of methylated library exceeds 70% |
Increase the ratio of unmethylated library. The ratio of unmethylated library sequenced in each lane should be > 30%.
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| Possible Reason | Solution |
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Contamination of post-converted library a. The BS module is out of date. b. The oxidation-caused crystallization of bisulfite may significantly inhibit the conversion efficiency. Input DNA is too much. . |
Use filter tips. Replace the gloves and clean experiment table after the conversion process. a. Use the BS module within the period of validity. b. Minimize the exposure to air. For EM module, the input DNA should be under 200 ng.
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| Possible Reason | Solution |
|
Inaccurate quantification of input DNA
a. Low input DNA with BS module. |
The Qubit fluorometer is recommended for the quantification of fragmented DNA. Increase PCR cycles; Convert low input DNA with EM module. |
If the PCR tube did not cool down quickly after the denaturation procedure, the circularization efficiency may be very poor. During the operation, the PCR tube must be placed in an ice bath immediately.
Circularization efficiency = (total amount of ssCirDNA×2)/total amount of input dsDNA. The cyclization efficiency of this kit is between 20%-40% commonly.
This kit has been optimized to be compatible with circularization of libraries with both SI adapters and DI adapters on MGI platform.
This kit has good compatibility with libraries of different sample types and size. It can be applied for circularization of most types of libraries, such as gDNA, cfDNA, multiplex PCR products, FFPE DNA, methylated DNA and RNA libraries.
|
Possible Reason |
Solution |
|
The DNA sample contains more than 1 mM of EDTA |
Purify the DNA by using magnetic beads or spin columns,and dissolve the purified DNA with TE Solution. |
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The DNA sample contains impurities |
High quality DNA input, with OD260 /OD280 =1.8-2.0 and OD260 /OD230 =2.0-2.5. The residual guanidine salt and proteins may suppress the fragmentase activity. Purify the DNA by using magnetic beads or spin columns,and dissolve the purified DNA with TE Solution. |
|
Fail to thoroughly mix the reaction mix |
Mix thoroughly and centrifuge to collect the contents to the bottom of the tube before initiate the reaction. |
|
Possible Reason |
Solution |
|
The DNA is seriously degraded |
Degraded DNA, such as FFPE C DNA, may result in short fragments (150-200 bp) without change of the same fragmentation condition. |
|
The fragmentation process lasts too long |
Avoid operating multiple samples at one time. |
|
The operation under room temperature lasts too long |
Ensure all the processes operated on ice beforeloading into the thermo cycler. |
Possible Reason：Inappropriate dilution of adapters
Solution：For low DNA input, adapters should be diluted as recommended prior to use.