The Nextera Mate Pair protocol has been validated with the S-series Covaris sonicators. According to Covaris, the performance of the E-series (E210 and E220) should be similar when using the equivalent settings (if using the E210, follow the settings recommended for the S2; if using the E220, follow the settings for the S220). However, these settings might not be directly transferable and using a Covaris outside of the S-series as recommended in the protocol will likely require optimization.
The recommended input for the Gel-Free protocol is 1 μg genomic DNA to generate 48 libraries from 48 samples. When following the Gel-Plus protocol, use 4 μg genomic DNA as input. This amount prepares either 12 libraries with size-selections per sample or up to 48 libraries with as man as four size selections from each of the 12 inputs.
Yes, although the tagmentation reaction may generate a distribution of fragments with an apparent average insert size of 5 kb or more, the smaller fragments present circularize far more efficiently than the larger fragments. Therefore the average fragment size as detected in the sequencing data can be considerably smaller than suggested by the Bioanalyzer electropherogram of the tagmentation reaction.
The Illumina Nextera Mate Pair Library Preparation Kit produces libraries with a broad size range distribution, typically between 300–1500 bp, with a peak centered around 600–700 bp. It is not necessary to further size-select the library for sequencing purposes. Although the largest fragments may not cluster and sequence as efficiently, their presence in the library is not detrimental.
The Nextera Mate Pair Gel-Plus protocol with size-selection aims to generate mate pair libraries with a narrower size distribution and larger median fragment size. This is achieved by using a gel-based size-selection process to select DNA of a desired size range. A Mate Pair library with a narrow distribution of fragments will facilitate accurate structural variation detection. It will also help de novo assemblies of both simple and complex genomes by providing paired read information which spans larger repeat regions.
However, the degree of difficulty in generating gel-plus libraries increases as the length of fragments increases. Compared to libraries with smaller Mate Pair fragment sizes, libraries with larger fragment lengths are expected to have a lower final library yield and lower library diversity and are more challenging to make robustly. To increase the robustness of library generation when carrying out the Gel-Plus procedure, several steps in the protocol have been optimized and guidelines are provided.
The Nextera Mate Pair Gel-Free protocol is a low input (1 ug), simple, and robust protocol generating Mate Pair libraries with a broad distribution of fragment sizes, from 1.5 kb up to 15 kb with a median insert size of around 3 kb. The Gel-Free Protocol reliably yields high diversity libraries with fewer duplicates, allowing deeper sequencing of the library. Possible applications of gel-free Mate Pair libraries are de novo assembly (especially of smaller genomes) and structural variation detection studies. Although currently not supported by Illumina, the gel-free protocol may also be adapted for automation.
AMPure purification procedures have been validated by Illumina using the Axygen Maxymum Recovery 1.7 ml microcentrifuge tubes and the Invitrogen magnetic stand (part # CS15000) as specified in the Consumables and Equipment list in the User Guide. Comparable performance, such as yield and size distribution of the libraries, is not guaranteed when using other microcentrifuge tubes/tube formats or other magnets, and the protocol may take longer.
See Data Processing of Nextera Mate Pair Reads on Illumina Sequencing Platforms for the Mate Pair junction sequence. The document also contains information on how to perform adapter trimming and analysis of Mate Pair data sets.
The Nextera Mate Pair Kit employs both Nextera and TruSeq technologies. The input gDNA is initially tagmented with a Nextera transposome enzyme, which adds a Mate Pair junction adapter to the ends of the DNA molecules.
However, after circularization, the DNA is fragmented for a second time and TruSeq adapters are added. The subsequent PCR, clustering, and sequencing steps use the TruSeq adapters.
Thus, for sequencing purposes, completed Nextera Mate Pair libraries are considered to be TruSeq DNA libraries and must be sequenced using TruSeq sequencing primers and protocols.