Active Motif,
Tools to analyze nuclear function,
Your CartYour Cart 0 items

STAY INFORMED

Sign up to receive new product updates
and promotional pricing.

Next Gen DNA Library Kit

high complexity NGS libraries containing MIDs

The Next Gen DNA Library Kit is designed to prepare Next generation sequencing (NGS) libraries from double-stranded input DNA for use with Illumina® sequencing platforms. Libraries can be generated from as little as 10 pg high quality genomic DNA, ChIP DNA, FFPE DNA or cell-free DNA (cfDNA), which is 500-fold more sensitive than other library preparation kits. Additionally, libraries can be prepared from 100 ng DNA if preparing PCR-free libraries.

Schematic of MID
Schematic of a completed MID tagged and indexed DNA library molecule.

Next Gen DNA Library Kit is used in combination with the Next Gen Indexing Kit, which contains 16 unique index adapters that may be used individually, or in combination to multiplex samples together during cluster generation for co-sequencing on the same Illumina flow cell. The advantage of the Next Gen DNA Library preparation is the inclusion of molecular identifiers (MIDs) to enable accurate removal of PCR duplicates from single read sequencing. This helps to increase the number of unique alignments for more accurate data sets.

For more information please click on the Overview tab below. To download the protocol, please select the Documents tab.

Active Motif's Next Gen DNA Library Kit and Next Gen Indexing Kit are powered by Swift Biosciences.

Swift Biosciences logo
 
Name Format Cat No. Price  
Next Gen DNA Library Kit 16 rxns 53216 $505 Buy Now
Next Gen Indexing Kit 64 rxns 53264 $250 Buy Now

Next Gen DNA Library Kit Highlights

  • NGS libraries for use with Illumina platforms (MiSeq, HiSeq 2500, NextSeq 500)
  • Works with double-stranded input DNA including:  genomic DNA, ChIP DNA, FFPE DNA and cell-free DNA (cfDNA)
  • Use as little as 10 pg DNA (500-fold less input material required than other kits)
  • Prepare PCR-free libraries with only 100 ng DNA
  • Indexing kit contains 16 unique indices that can be multiplexed together within the same sequencing flow cell
  • Compatible with Active Motif's ChIP-IT High Sensitivity® Kit for a complete ChIP-seq workflow

 

How Does the Next Gen DNA Library Kit work?

The Next Gen DNA Library Kit is designed to generate high complexity DNA libraries for Next generation sequencing (NGS). The kit offers the advantage of including molecular identifiers (MIDs) during library generation to enable removal of true PCR duplicates from the sequencing analysis. This helps to disntinguish PCR duplicates from fragmentation duplicates, thereby, increasing the number of unique alignments for more accurate data analysis. 

Next Gen DNA Library Kit flow chart
Next Gen DNA Library Kit flow chart.

 

  • Using four incubations, this protocol repairs both 5´ and 3´ termini and sequentially attaches Illumina adapter sequences to the ends of fragmented dsDNA.
  • Bead-based SPRI clean-ups are used to remove oligonucleotides and small fragments and to change enzymatic buffer composition between steps. Different SPRIselect bead-to-sample ratios are utilized for different input quantities and insert sizes.
  • For PCR-free applications, the resulting functional library is ready for library quantification and sequencing on the Illumina platform.
  • Alternatively, an optional PCR step may be used to increase yield of indexed libraries, which then may be quantified and sequenced.

Incorporation of Molecular Identifiers (MIDs)

During Next Gen DNA Library generation, standard low throughput (LT) P7 adapters containing a single index for multiplexed sequencing (index 1 position) during Ligation I. Following a PEG NaCl clean-up, a high throughput (HT) P5 adapter containing the MID is incorporated during Ligation II. The MID is a 9 base random N sequence at the [i5] (index 2 position) of the P5 adapter. Addition of the MID is strand-specific with each dsDNA insert receiving two MIDs (X and Y). The two MIDs cluster and sequence independently. Bioinformatically, PCR replicates can be removed from the data set, while fragmentation replicates are preserved to increase the number of unique alignments and improve data analysis.

Schematic of MID

Kit Performance

The Next Gen DNA Library Kit (Catalog No. 53216) can be used to prepare high quality NGS libraries from dsDNA. It is designed to be used in combination with Active Motif's Next Gen Indexing Kit (Catalog No. 53264). Both kits are required to prepare the NGS libraries.

Input requirements:  We recommend using between 10 pg - 250 ng input DNA per library preparation. Input should be fragmented double-stranded DNA. For PCR-free library preparation a minimum of 100 ng DNA is needed. For library amplification by PCR, as little as 10 pg DNA is required. 

Expected yield:  Yields will vary between sample types and input amounts, but > 4 nM is expected.

 

Next Gen DNA Library Kit Prepares High Quality ChIP-seq Libraries

 

ChIP-Seq data
Figure 1: Next Gen DNA Library Kit prepares high quality ChIP-seq libraries, even with lower cell numbers.

Active Motif's Low Cell ChIP-Seq protocol was used to prepare chromatin from GM12878 cells using the number of cell equivalents listed. ChIP-seq libraries were prepared using either the Next Gen DNA Library Kit or a competitor kit. Results show strong peaks that match ENCODE data sets for the Next Gen Libraries, even if starting from low cell numbers.

Contents & Storage

Please note that the Next Gen DNA Library and Next Gen Indexing Kits are shipped on dry ice and contains reagents with multiple storage temperatures inside. All components can be stored at -20°C prior to first use, then we recommend storing each component at the temperatures indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Each kit includes the following components:

Reagents for Next Gen DNA Library Kit (Cat. No. 53216)

  • Buffer W1; Store at -20°C
  • Enzyme W2; Store at -20°C
  • Buffer G1; Store at -20°C
  • Reagent G2; Store at -20°C
  • Enzyme G3; Store at -20°C
  • Enzyme G4; Store at -20°C
  • Buffer Y1; Store at -20°C
  • Enzyme Y3 Store at -20°C
  • Buffer B1; Store at -20°C
  • Reagent B3; Store at -20°C
  • Enzyme B4; Store at -20°C
  • Enzyme B5; Store at -20°C
  • Enzymbe B6; Store at -20°C
  • Reagent R2; Store at -20°C
  • Buffer R3; Store at -20°C
  • Enzyme R4; Store at -20°C
  • Alu 115; Store at -20°C
  • Alu 247; Store at -20°C
  • PEG Solution; Store at RT
  • Low EDTA TE; Store at RT

Reagents for Next Gen Indexing Kit (Cat. No. 53264)

  • Reagent Y2 Index 1; Store at -20°C
  • Reagent Y2 Index 2; Store at -20°C
  • Reagent Y2 Index 3; Store at -20°C
  • Reagent Y2 Index 4; Store at -20°C
  • Reagent Y2 Index 5; Store at -20°C
  • Reagent Y2 Index 6; Store at -20°C
  • Reagent Y2 Index 7; Store at -20°C
  • Reagent Y2 Index 8; Store at -20°C
  • Reagent Y2 Index 9; Store at -20°C
  • Reagent Y2 Index 12; Store at -20°C
  • Reagent Y2 Index 13; Store at -20°C
  • Reagent Y2 Index 14; Store at -20°C
  • Reagent Y2 Index 15; Store at -20°C
  • Reagent Y2 Index 16; Store at -20°C
  • Reagent Y2 Index 18; Store at -20°C
  • Reagent Y2 Index 19; Store at -20°C
  • Reagent R1; Store at -20°C
  • Reagent B2 MID; Store at -20°C