Input Requirements

Input Requirements

The Nextera Mate Pair Sample Preparation Kit protocol is optimized for 1 μg of genomic DNA if performing the Gel-Free protocol or 4 μg of DNA if performing the Gel-Plus protocol. Illumina strongly recommends quantifying the starting genomic material.

Input DNA Quantitation

Nextera Mate Pair Sample Preparation kit uses an enzymatic DNA fragmentation step and is more sensitive to DNA input than mechanical fragmentation methods. The ultimate success of library preparation strongly depends on using an accurate amount of input genomic DNA. Therefore, the correct quantitation of the input DNA sample is essential. To obtain an accurate quantification of the genomic DNA, it is recommended to quantify it using a fluorometric based method specific for duplex DNA such as the Qubit dsDNA BR Assay system. Illumina recommends using 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Methods that measure total nucleic acid content (e.g. nanodrop or other UV absorbance methods) should be avoided because common contaminants such as ssDNA, RNA, and oligos are not substrates for the Nextera Mate Pair Sample Preparation protocol.

Assessing DNA Quality

Use of high-quality, high molecular weight genomic starting material is also important for successful library generation. If degraded genomic DNA is used, the tagmentation step of the protocol is likely to fragment the DNA to a size below the desired size range. Furthermore, damaged and degraded DNA samples will PCR amplify less efficiently, leading to diminished library yields and diversity

A simple way to assess the quality of the starting material is to run a small amount on a low-percentage agarose gel. High-quality DNA should run as a high molecular weight band with the majority of DNA greater than 50 kb in size and with minimal lower molecular weight smearing. If the majority of the DNA is below 50 kb or smearing is visible, this suggests that the DNA is degraded. If the sample is partially degraded, the Nextera Mate Pair Sample Preparation Protocol can still be used, but the amount of Transposome used in the tagmentation step may have to be reduced to prevent overtagmentation and generate DNA fragments in the desired size range.