Publications Placeholder

Next-generation Sequencing


An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage

Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive assessment of cancer burden, but existing ctDNA detection methods have insufficient sensitivity or patient coverage for broad clinical applicability. Here we introduce cancer personalized profiling by deep sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. We implemented CAPP-Seq for non–small-cell lung cancer (NSCLC) with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumors. We detected ctDNA in 100% of patients with stage II–IV NSCLC and in 50% of patients with stage I, with 96% specificity for mutant allele fractions down to ~0.02%. Levels of ctDNA were highly correlated with tumor volume and distinguished between residual disease and treatment-related imaging changes, and measurement of ctDNA levels allowed for earlier response assessment than radiographic approaches. Finally, we evaluated biopsy-free tumor screening and genotyping with CAPP-Seq. We envision that CAPP-Seq could be routinely applied clinically to detect and monitor diverse malignancies, thus facilitating personalized cancer therapy.

Aaron M Newman, Scott V Bratman, Jacqueline To, Jacob F Wynne, Neville C W Eclov, et al.
Nature Medicine


ChIP Sequencing

Dana-Farber Cancer Institute and Harvard Medical School

  • KAPA High-Throughput Library Preparation Kit

 

 

 

 

Automation of ChIP-Seq Library Preparation for Next Generation Sequencing on the epMotion® 5075 TMX

ChIP-Seq library preparation can be a challenging procedure to automate because of its low ChIP DNA input. This Application Note describes the successful automation of Illumina ChIP-Seq library preparation on the Eppendorf epMotion 5075 TMX, using the KAPA High-Throughput Library Preparation Kit from Kapa Biosystems. Size-selected libraries were prepared from as little as 1 ng of fragmented ChIP DNA. To obtain the recommended 100 ng of library material for sequencing on the Illumina Genome AnalyzerIIx, only 18 amplification cycles were needed for 1 ng of input DNA, or 9 cycles when starting library construction with 10 ng of ChIP DNA.DNA Library Preparation Cheng Liu Ph.D. and Jesse Cassidy, Eppendorf North America, Hauppauge, NY, USA Maryke Appel Ph.D., KAPA Biosystems, Inc., Woburn, MA, USA. Biotechniques

Cheng Liu Ph.D. and Jesse Cassidy, Eppendorf North America, Hauppauge, NY, USA
Maryke Appel Ph.D., KAPA Biosystems, Inc., Woburn, MA, USA


DNA Library Preparation

 

 

 

 

Characterizing and measuring bias in sequence data

DNA sequencing technologies deviate from the ideal uniform distribution of reads. These
biases impair scientific and medical applications. Accordingly, we have developed
computational methods for discovering, describing and measuring bias.

Michael G Ross§, Carsten Russ, Maura Costello, Andrew Hollinger, Niall J Lennon, Ryan
Hegarty, Chad Nusbaum, David B Jaffe
The Broad Institute, 7 Cambridge Center, Cambridge, MA 02142


Library Quantification

 

 

 

 

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels),copy number alterations and selected fusions across 287 cancer related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens.


Methyl Sequencing

 

 

 

 

Ultra-low input, tagmentation-based whole-genome bisulfite sequencing

We have adapted transposase-based in vitro shotgun library construction (‘‘tagmentation’’) for whole-genome bisulfite
sequencing. This method, Tn5mC-seq, enables a >100-fold reduction in starting material relative to conventional
protocols, such that we generate highly complex bisulfite sequencing libraries from as little as 10 ng of input DNA, and
ample useful sequences from 1 ng of input DNA. We demonstrate Tn5mC-seq by sequencing the methylome of a human
lymphoblastoid cell line to ~8.63 high-quality coverage of each strand.

Andrew Adey and Jay Shendure1
Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA

 

 

 

 

Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins


Multiplex

CTA_58

 

 

 

 


Sample QC

CTA_17

 

 

 

 

Importance of Reliable Quantification and Quality Assessment of Challenging FFPE Samples

Dr. Robert Daber discusses how the KAPA hgDNA Quantification and QC Kit improves clinical sequencing workflows.


qPCR

KAPA PROBE FAST


CTA_45

 

 

Molecular characterization of human T-cell lymphotropic virus type 1

Here, we report on the partial and full-length genomic (FLG) variability of HTLV-1 sequences from 90 well-characterized subjects, s, including 48 HTLV-1 asymptomatic carriers (ACs), 35 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and 7 adult T-cell leukemia/lymphoma (ATLL) patients, using an Illumina paired-end protocol.


KAPA2G Robust

CTA_46

 

 

Improved efficiency and robustness in qPCR and end-point PCR

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 59-end. In qPCR, the 59-o-TINA modified primers allow for a qPCR R efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 59-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 59-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased
annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches.

Gala2_mdIIn a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 59-o-TINA modified primers allows for a further reduction (.45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 59-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures
for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 59-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.

Schneider UV, Mikkelsen ND, Lindqvist A, Okkels LM, Jøhnk N, et al.
PLoS ONE 7(6): e38451. doi:10.1371/journal.pone.0038451

PCR

KAPA Blood

Express barcodes racing from specimen to identification

KAPA2G Fast

Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments

KAPA HiFi

Optimal enzymes for amplifying sequencing libraries

KAPA2G Fast Multiplex

Improving Lamda Red genome engineering in E. coli via rational removal of endogenous nucleases

KAPA3G Plant

Engineered DNA polymerase improves PCR results for plastid DNA

Publications

Nature Medicine

An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive assessment of cancer burden, but existing ctDNA detection methods have insufficient sensitivity or patient coverage for broad clinical applicability. Here we introduce cancer personalized profiling by deep sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. We implemented CAPP-Seq for non–small-cell lung cancer (NSCLC) with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumors. We detected ctDNA in 100% of patients with stage II–IV NSCLC and in 50% of patients with stage I, with 96% specificity for mutant allele fractions down to ~0.02%. Levels of ctDNA were highly correlated with tumor volume and distinguished between residual disease and treatment-related imaging changes, and measurement of ctDNA levels allowed for earlier response assessment than radiographic approaches. Finally, we evaluated biopsy-free tumor screening and genotyping with CAPP-Seq. We envision that CAPP-Seq could be routinely applied clinically to detect and monitor diverse malignancies, thus facilitating personalized cancer therapy.

Aaron M Newman, Scott V Bratman, Jacqueline To, Jacob F Wynne, Neville C W Eclov, et al.
Nature Medicine

  • KAPA High-Throughput Library Preparation Kit
  • Keyword 2