CleanPlex® Targeted Library Kit and CleanPlex® custom panels are used together to amplify target regions of DNA using multiplex PCR for Next-Generation Sequencing (amplicon sequencing) target enrichment and library preparation on Illumina Sequencers.
CleanPlex® Targeted Library Kit
CleanPlex® Targeted Library Kit and CleanPlex® custom panels are used together to amplify target regions of DNA and construct targeted libraries for Next-Generation Sequencing (amplicon sequencing) on Illumina Sequencers. The amplification products (including target amplicons and nonspecific products) from CleanPlex® multiplex PCR reactions are purified and treated with CleanPlex® Digestion Reagent to degrade the remaining nonspecific PCR products. The amplicons are further purified with magnetic beads and re-amplified with primers containing indexes (“index primers”) to form an amplicon DNA library.
Please note that ready-to-use panel kits such as CleanPlex® OncoZoom Panel and CleanPlex® BRCA1 & BRCA2 Panel already contain all the reagent components of CleanPlex® Targeted Library Kit.
When multiple libraries are made and each library is attached with a unique index, these libraries may be combined (pooled) in a variety of ways before being sequenced on Illumina Sequencers. Combining libraries maximizes the usage of flow-cell while minimizing cost and labor.
Before sequencing, the final library (either a single library or a pool of libraries each with a unique index) can be quantified by qPCR, or by a method compatible with Illumina Sequencers.
- Easy integration with liquid handlers and automation devices for high-throughput NGS labs. 2.5-hour and 3-step target enrichment workflow with only 30-minute hands-on time from purified DNA to sequencing-ready library.
- Save time, labor, reagents, and capital equipment. NO DNA fragmentation, NO overnight hybridization, NO microfluidic device, NO ligation, NO end repair anymore!
- Save your precious samples. Only 100 pg of input DNA is needed for germline genotype calling and 10 ng for somatic mutation calling with an LOD of 1%.
- Accurate variant calling. High uniformity and on-target rate lead to highly sensitive and specific NGS variant calls.
- Highly scalable multiplex PCR accommodates your expanding panel needs. 7 – 20,000 PCR reactions per tube plus pooling strategy can meet the needs of most applications.
- Enable new applications. Able to amplify difficult or high GC genomic regions such as TERT promoter (involved in many types of human cancers) – this is included the CleanPlex® OncoZoom Cancer Panel, a 65-gene cancer hotspot panel.
- Save oligo synthesis cost. No chemical modification of primers lowers your oligo synthesis costs by 4-10 fold.
|Dimensions||12 x 10 x 9 cm|
|Pack Size (Reactions)|
8, 96, 384, 1152, 4608
(1) 30% ethanol instead of 70% ethanol is used in DNA purification with magnetic beads; or
(2) forgetting to add magnetic beads after digestion. The workflow does not require users to change tubes, nor to remove magnetic beads from previous steps. One can easily tell if magnetic beads in each purification steps are added or not by checking the volume; or
(3) forgetting to add Stop Buffer after digestion, especially when handling a large number of samples.
(4) over-drying beads can decrease library yield, whereas under-drying beads can leave ethanol that will inhibit PCR.
(5) inaccurate volume of magnetic beads added. Ensure magnetic bead aliquot is well mixed prior to each dispense.
(6) insufficient mixing. Ensure all reactions and solutions are thoroughly mixed and homogeneous prior to proceeding to thermocycling and incubation steps.
There are peaks from 70 – 90 bp in addition to the main library peak on the Agilent™ 2100 Bioanalyzer chart?
There is a peak around 150 bp in addition to the main library peak on the Agilent™ 2100 Bioanalyzer chart.
Significant peaks from 150 – 160 bp may be caused by under-amplification of the library (see the graph below on the right). Certain panels may tend to produce more non-specific amplification products. In this case, two rounds of magnetic bead purification after the digestion step solves the problem.