FFPE Sample NGS Library Preparation Attention Details

FFPE Sample NGS Library Preparation Attention Details

DNA Quality Control

DNA quality control is required before library construction to check the total amount (concentration), integrity and purity of the DNA, which can affect the success rate of library preparation and the performance of subsequent sequencing data due to insufficient initial DNA input, DNA degradation and contamination by impurities. Therefore, concentration determination must be accurate in order to ensure the accuracy of the library input. If there is degradation of DNA by electrophoresis, the interruption time of subsequent DNA needs to be adjusted appropriately according to the degree of degradation. If there is protein, RNA and other impurities contamination, it needs to be digested and purified with proteinase K, RNAase, etc. The purpose is to know the quality of DNA before library preparation, so that on the one hand, we can take measures in advance to improve the success rate of library preparation, and on the other hand, we can better trace the cause when there are problems with subsequent experiments or sequencing data indicators.

Damage repair before breakage

FFPE samples can make some degree of damage to genomic DNA during preparation and preservation, such as gaps, cytosine deamidation to uracil, etc. To improve library quality, it is recommended that DNA be repaired with a specific enzyme mixture before interruption.

Interruption time

FFPE samples are subject to different degrees of DNA degradation when extracted due to the preservation environment as well as time. So when interrupting, the number of interrupting cycles (i.e. interruption time) needs to be adjusted according to the degree of DNA degradation so that the interrupted fragment peaks are concentrated as much as possible within the expected range.

The amount and type of variation in DNA damage, if ignored, may have a negative impact on the final result, which can have a huge impact on downstream applications such as sequencing: from the failure of simple sequencing library preparation to the generation of spurious libraries, ultimately leading to incorrect results.