Questions & Answers

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General

  • Can I use a Covaris E210 or E220 instead of the S2 or S220 sonicators?

    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, please note that these settings may not be directly transferable and using a Covaris outside of the S-series as recommended in the protocol will likely require optimization.

  • What is the input amount for each protocol and how many samples can I prepare using the Nextera Mate Pair kit?

    When following the Gel-Free protocol, the recommended input is 1 μg of genomic DNA and the kit is formulated to generate 48 libraries from 48 independent samples. When following the Gel-Plus protocol, the recommended input is 4 μg of genomic DNA and this kit is formulated to either prepare 12 libraries with single size-selections per sample, or up to 48 libraries if making as many as 4 size-selections from each 12 inputs.

  • The distribution of fragment sizes seen after tagmentation is much larger than the size distribution of Mate Pair reads seen in my sequencing data. Is this expected?

    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.

  • According to my BioAnalayzer traces, my final library has a size distribution of 300 to 1500 bp. Will these large-sized fragments sequence? Should I perform further size selection on these libraries?

    The Illumina Nextera Mate Pair Sample Preparation kit produces libraries with a broad size range distribution, typically between 300-1500 bp, with a peak centred around 600-700 bp. It is not necessary to size select the library further for sequencing purposes. Although the largest fragments may not cluster and sequence as efficiently, their presence in the library is not detrimental.

  • What are the advantages of the Gel-Plus Protocol?

    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.

  • What is new about the Nextera Mate Pair Protocol as compared to the Illumina v2 Mate Pair protocol?

    The new Nextera Mate Pair kit offers several advantages over the previous Mate Pair kit: significantly less DNA input (1-4 ug as compared to 10-20 ug for the original kit), a faster and simplified workflow, and a higher supported read length of 2x250.

    Other advantages of the Nextera Mate Pair kit include utilization of a transposome-mediated fragmentation and adapter tagging of genomic DNA, an identifiable Mate Pair junction sequence, TruSeq DNA Sample Prep master-mixed reagents to reduce reagent containers and pipetting steps, TruSeq DNA Sample Prep adapter indexing compatibility with 12 indexes included in the kit, and a choice of two protocols: a Gel-Free Protocol and a Gel-Plus Protocol.

  • What are the advantages of the Gel-Free Protocol?

    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.

  • Can I use a different magnet or tube when handling the AMPure beads other than the ones recommended in the User Guide?

    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.

  • What is the sequence of the Mate Pair junction adapter?

    Please refer to the document Data Processing of Nextera Mate Pair Reads on Illumina Sequencing Platforms for the Mate Pair junction sequence. This document also contains additional information on how best to perform adapter trimming and analysis of Nextera Mate Pair data sets.

  • Which sequencing primers do I use for sequencing Nextera Mate Pair libraries: Nextera or TruSeq?

    For sequencing purposes, the final completed Nextera Mate Pair libraries are technically considered to be TruSeq DNA libraries, and should be sequenced with TruSeq sequencing primers and workflows. The Nextera Mate Pair Kit actually employs both Nextera and TruSeq technologies. The genomic DNA 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, at which point the TruSeq adapters are added. It is the TruSeq adapters that are utilized during the subsequent PCR, clustering and sequencing steps.