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.
- 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.
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