The NxSeq® UltraLow DNA Library Kit, 96 Reactions and NxSeq® HT Dual Indexing Kit share all the same features and performance characteristics as the smaller, NxSeq UltraLow DNA Library Kit, 12 Reactions and Single Indexing Kits. This 96 reaction kit is formatted for the construction of DNA fragment libraries in 96-well plates. The kit can be used with multichannel pipettes or with automated liquid-handling systems. To facilitate 96-well library prep, we’ve provided sufficient overfill of each reagent and a detailed, step-by-step protocol in the User Manual.
To aid in accurate dispensing of the NxSeq HT Dual Indexing Primers across a 96-well PCR plate, we added a different inert dye to each set of primers. The 700 Series Indexing Primers contain a blue dye, while the 500 Series Indexing Primers contain an orange/yellow dye. With colored primer sets, you can easily see when each primer is added to the correct row or column of the 96-well PCR plate and verify that all primers are correctly dispensed (Figure 2). We performed extensive testing to verify that these dyes do not interfere with the Illumina instrument or sequencing results. See Figure 3 below and our FAQs for detailed information.
Key features shared between the 96 and 12 reaction kits include: (i) ability to build highly complex DNA fragment libraries from as little as 50 pg of DNA, (ii) easy workflows with a minimal number of reagents, (iii) improved coverage and better depth uniformity, and (iv) low library prep costs with high data quality. Both kits and protocols produce identical, high quality sequencing data. Please visit the NxSeq UltraLow DNA Library Kit, 12 Reactions and Single Indexing Kits webpage for additional performance data.
Both NxSeq UltraLow DNA Library and Indexing Kits were optimized for whole genome sequencing (WGS) and resequencing for de novo sequencing or SNV/mutation or copy number variation (CNV) identification. Both systems, however, are also compatible with other applications such as ChIP-seq, exome-seq, and with other sample types such as FFPE and metagenomic DNA samples.
See the FAQs for more information on these kits.
Figure 1. Mechanics of NxSeq® UltraLow Kit library construction using dual indexing primers. This figure illustrates how DNA fragment libraries are constructed using the NxSeq® UltraLow Library Prep Kit and the NxSeq® HT Dual Indexing Kit. Each final library fragment has a 500 Series Index at one end and a 700 Series Index at the other end. Note that some PCR fragments and products are not shown to simplify the illustration.
Figure 2. 96-well PCR plate dual index primer dispensing protocol. The 700 Series Indexing Primers contain an inert blue dye and the 500 Series Indexing Primers contain an inert yellow/orange dye. The 700 Series Primers are dispensed first, row by row. As primers are dispensed, the rows become blue. The 500 Series Primers are then dispensed column by column, and as they are added the well color changes from blue to green (blue + yellow = green). After dispensing is complete, all wells should exhibit a consistent green color. The presence of clear, yellow/orange or blue wells indicates that those wells did not get the appropriate 700 and/or 500 Series Primers.
|Average Number of Reads||145,192,042||149,394,251|
|Percent Mapped Reads||95.4%||95.4%|
|Insert Length||258 bp||255 bp|
|Proper Pair Duplication Rate||8.4%||8.8%|
Figure 3. Analyzing the effect of the inert dyes on sequencing results. Sheared human gDNA was generated by mechanical shearing (Covaris LE220, peak size 300 bp) and six libraries were constructed using 10 ng of sheared gDNA input per library and the NxSeq® UltraLow DNA Library Kit, 96 Reactions according to the User Manual up through Universal Adaptor Ligation and Bead Cleanup steps. After cleanup, the 6 adaptor-ligated libraries were pooled, mixed and split into six PCR reactions. The two inert dyes present in the NxSeq® HT Dual Indexing Primers were then added to three PCR reactions with each dye at the same concentration as in PCR reactions set up using the NxSeq HT Dual Indexing Primers. An equivalent volume of water was added to the other three PCR reactions. Indexing primers were added to each reaction (different indices) followed by PCR amplification of all libraries, library Cleanup and Size Selection. The final libraries were sent to Hudson Alpha for sequencing on the HiSeq X Ten. Note that ExAmp duplicates caused by the instrument were not removed bioinformatically. The results presented are the average results for each set of triplicate libraries.
|gDNA Input per |
|Coeffient of Variance (CV) of |
Library Yield per Set of
|Average CV by Organism|| |
|Average CV for Plate||11.1%|
Figure 4. Analysis of variation in library yield for libraries generated in a 96-well plate using the multichannel pipette protocol. Sheared gDNA was generated by mechanical shearing (Covaris LE220, peak size 300 bp) for the three genomes indicated. The indicated amounts of each gDNA were dispensed into a 96-well plate with four replicates of each input amount per organism and libraries were constructed using the NxSeq® UltraLow DNA Library Kit, 96 Reactions and the NxSeq® HT Dual Indexing Kit according to the User Manual (MA169). Note that the libraries had to be removed from the plate for PCR amplification due to the different number of PCR cycles per gDNA input amount, but all other steps including AMPure XP bead cleanup and size selection steps were performed in a single 96-well plate. Final libraries were quantitated on a Qubit® Fluorometer and the coefficient of variation (CV %) for each set of replicates was determined. The average CV% by organism (columns) and across the entire plate (last row) were also determined. Note that there were four outliers that significantly increased the already low plate CV and by removing them from the analysis the CV drops to 8%.
|Library 1||Library 2|
|Total PF Sampled Reads||266,008,126||266,337,140|
|Fragment Length (bp)||314 +/- 90||321 +/- 92|
|Duplicates (Proper Read Pairs)||7.64%||6.12%|
|Coverage Depth||9.93 +/- 5.12||10.35 +/- 5.45|
Figure 5. HiSeq 2500 sequencing of two human libraries prepared in a 96-well plate using the multi-channel pipette protocol. Two of the human 2 ng libraries prepped in the Figure 3 experiment were sequenced on a HiSeq 2500 using 2 x 150 bp chemistry and analyzed.