Application Notes

DNA Shearing for Next Generation Sequencing (NGS) with the M220 Focused-ultrasonicator

Application Notes

DNA Shearing for NGS with the ME220 Focused-ultrasonicator.

Lysis and High Molecular Weight DNA Extraction from Eukaryotic and Prokaryotic Cells Powered by AFA-energetics®

Application Notes

This application note describes an automatable approach to cell lysis and extraction of high molecular weight DNA from microbial samples, which can be sequenced with long-read sequencing technology.

Lysis and Extraction of Biomolecules from Eukaryotic and Prokaryotic Cells Powered by AFA-energetics®

Application Notes

Covaris developed a novel automatable approach for the extraction of biomolecules from microbial samples for analysis with mass spectrometry technologies using Adaptive Focused Acoustics® technology.

Automated Sample Preparation Combining Adaptive Focused Acoustics® (AFA®) and Single Pot Solid Phase Sample Preparation (SP3)

Application Notes

Automated Protein Extraction from Clinical Samples with the Covaris ML230 Focused-ultrasonicator

Application Notes

Hands-off Sample Preparation Workflow for Laser Capture Microdissection (LCM) Samples using Adaptive Focused Acoustics (AFA)

Application Notes

Active paraffin removal from FFPE tissues with Adaptive Focused Acoustics

Application Notes

Covaris developed a method of active paraffin removal from FFPE tissues using AFA technology. AFA treatment is applied to a FFPE sample and paraffin is separated from the tissue by emulsification.

Automated Clinical Rapid Whole Exome Sequencing with the Covaris R230 Focused-ultrasonicator

Application Notes

Covaris Technical Note with Radboudumc

DNA fragmentation is a critical step in the preparation of good quality libraries for next-generation sequencing (NGS). Covaris Adaptive Focused Acoustics^® (AFA^®) technology remains the gold standard in reproducible and robust fragmentation, especially relevant while working with precious clinical samples.

Active Extraction of Native Proteins from Yeast using Covaris Adaptive Focused Acoustics® (AFA®)

Application Notes

Native protein extraction reagents were tested for total protein yield and preservation of enzymatic activity using a “passive extraction” or an AFA-based “active extraction” method, both increased.

AFA-sonication Followed by Modified Protein Aggregation Capture (APAC) Enables Direct, Reproducible and Non-toxic Sample Preparation of FFPE Tissue for Mass Spectrometry-based Proteomics

Application Notes

The preparation of formalin-fixed and paraffin-embedded (FFPE) tissue for mass spectrometry-based (MS) proteomics relies on efficient removal of paraffin, which commonly involves toxic xylol as gold standard.

Analysis of DNA fragments using the Agilent 2100 Bioanalyzer

Application Notes

DNA shearing protocols for Focused-ultrasonicators with the Agilent DNA 12000 kit was used to evaluate the average base pair size because of the large fragment size range, from 130 bp to 5 kb range.

Covaris DNA shearing guide for the Agilent SureSelect Target Enrichment System©

Application Notes

DNA Shearing

Optimizing Sample Fixation and Chromatin Shearing for Improved Sensitivity and Reproducibility of Chromatin Immunoprecipitation

Application Notes

Adaptive Focused Acoustic (AFA) technology provides precise control over mechanical shearing and thermal control during processing to deliver high quality chromatin for sensitive results.

DNA Extraction from FFPE samples with Covaris Focused-ultrasonicators

Application Notes

When an Adaptive Focused Acoustics (AFA) treatment is applied to a FFPE sample to extract DNA, it simultaneously deparaffinizes the sample, emulsifies the paraffin, and rehydrates the tissue.

Robust Sample Preparation for Label-free or Tandem Mass Tag (TMT) LC-MS with Adaptive Focused Acoustics® (AFA®)

Application Notes

Variability in DNA Fragment Size and Distribution Analysis Across Various Fragment Analyzers

Application Notes

DNA Fragmentation

Impact of AFA-energetics™ on Enzymatic Hydrolysis of Proteins

Application Notes

A novel fully automatable approach to improve trypsin digestion of protein samples is presented.

Clinical Tumor Tissue Evaluated with Illumina® TruSight™ Oncology 500 (NGS) Assay and Sheared on the Covaris ML230 Focused-ultrasonicator

Application Notes

Abstract: DNA fragmentation is a critical step in the preparation of high-quality next generation sequencing (NGS) libraries. Covaris Adaptive Focused Acoustics® (AFA®) technology guarantees highly reproducible fragmentation of nucleic acids, which is especially relevant while working with valuable clinical samples for pan-cancer NGS assays such as the Illumina®’s TruSight™ Oncology 500 (TSO 500) panel. This panel was developed jointly by Illumina® and Covaris to provide high quality sequencing results. The DNA fragmentation protocol was previously released for the Covaris E220evolution, LE220-plus, and ME220 Focused-ultrasonicators [1]. In this application note, we present the ML230 and microTUBE-50 protocol to shear DNA to a fragment distribution optimized for the TSO 500 panel.

Optimized Covaris truXTRAC® FFPE RNA Protocol for Isolating RNA from Laser-Capture Microdissected (LCM) FFPE Tissue for Next Generation Sequencing

Application Notes

Covaris truXTRAC FFPE RNA kits and Focused-ultrasonicators enables NGS-grade nucleic acids for RNA sequencing, a feat previously challenging when using low input FFPE tissue such LCM-FFPE samples.

A Comparison of Active and Passive FFPE DNA Extraction Methods and Effects on Downstream Analysis Metrics

Application Notes

Active and Passive FFPE DNA Extraction

Amplicon Shearing Using a Covaris® Focused-ultrasonicator

Application Notes

In this application note, we describe the use of Covaris AFA® technology for mechanical DNA shearing of amplicons ranging in size in preparation for massively parallel high-throughput sequencing.

Protein Extraction from Yeast: Comparison of the Covaris Adaptive Focused Acoustics®(AFA) Process to Conventional Bead Beating and Probe Sonication

Application Notes

The efficiency of several mechanical-based lysis and extraction techniques, such as Adaptive Focused Acoustics (AFA), probe sonication, and bead beating from yeast isolates was compared.

A Robust and Standardized Workflow to Analyze C. albicans Differential Protein Expression using Tandem Mass Tag (TMT)

Application Notes

A Streamlined Workflow to Perform ChIP from FFPE Samples

Application Notes

We demonstrate how AFA® technology combined with truXTRAC® and truChIP® effectively recovers chromatin suitable for studying histone modifications and transcription factor binding interaction events.

Optimizing a Dual Fixation Protocol to Study Protein Complexes Binding to Chromatin in vivo

Application Notes

This application note provides shearing profiles, evaluates epitope integrity each shearing time and ChIP-qPCR results obtained using single step formaldehyde fixation and dual crosslinking protocol.

High-Throughput, Low Volume gDNA Extraction from Whole Blood Enabled by Covaris Adaptive Focused Acoustics® (AFA®) and AFA-TUBE® TPX

Application Notes

Extraction of gDNA from whole blood is the first step in multiple translational research and molecular diagnostics applications, such as next-generation sequencing, multiplex PCR, qPCR, and ddPCR.

truXTRAC® FFPE RNA Extraction and Purification - Quality Metrics for Clinical Applications Powered by Adaptive Focused Acoustics® (AFA®)

Application Notes

FFPE RNA Extraction

Nuclei EXtraction by SONication - NEXSON

Application Notes

NEXSON

Tumor Tissue Evaluation with the Illumina® TruSight™ Oncology 500 (NGS) Assay using the Covaris R230 Focused-ultrasonicator for Clinical Research Studies

Application Notes

Abstract: Next-generation sequencing (NGS) library preparation workflows comprise DNA fragmentation as an essential step. Covaris’ Adaptive Focused Acoustics® (AFA®) Technology enables precise and high-quality data from NGS workflows by ensuring efficient, reliable, and reproducible nucleic acid fragmentation. Efficient DNA shearing is particularly important when working with precious clinical research samples for Comprehensive Cancer Profiling (CCP) using pan-cancer NGS assays such as Illumina’s TruSight Oncology 500 (TSO 500) panel. The DNA fragmentation protocol for the TSO 500 panel was initially released for the Covaris Focused-ultrasonicators ME220, E220evolution, LE220, and ML230 (1,2).In this application note, we present a DNA fragmentation protocol with the R230 Focused-ultrasonicator using 96 microTUBE plates to achieve the optimal fragment size for the TSO 500 panel. A comparative sequencing analysis between samples sheared with the Illumina-validated protocol on the LE220 focused-ultrasonicator and those sheared using the Covaris developed protocol on the R230 showed equivalent or better results for samples sheared on the R230, while decreasing the sample processing time by ~30%. The results in this application note highlight that the automatable R230 enables efficient, high-throughput workflows with Covaris AFA Technology resulting in the robust, reproducible, and confident fragmentation needed for comprehensive pan-cancer panels such as Illumina´s TruSight Oncology 500 assay.

Release of Microbial Cells from a Solid Matrix, Cell Lysis, and Shearing of Released Nucleic Acids Performed in a One-Step Procedure

Application Notes

Release of Microbial Cells

High Quality DNA and RNA Extraction and Purification from FFPE Samples with Covaris truXTRAC® FFPE tNA Ultra Kit – Magnetic Beads

Application Notes

Streamlined Ultra Low Sample Input and Processing Volume Chromatin Shearing Protocols for Fly Embryos and Mammalian Cell Lines

Application Notes

These protocols have been optimized for both mammalian cell lines and fly embryos and reliably fragment chromatin in low volumes from down to 10,000 mammalian cells and 5 stage-17 Drosophila embryos.

Combining Covaris 96 AFA-TUBE TPX Plate with Swift Biosciences Accel-NGS 2S Kit for an Optimized Single Tube Workflow for Robust, Low Input NGS Library Preparation

Application Notes

AFA-TUBE Case study

Optimized Protocol for Robust Chromatin Shearing and Immunoprecipitation of Human Pancreatic Islets using the Covaris® Focused-ultrasonicator

Application Notes

We demonstrate how the Covaris S220 Focused-ultrasonicator provides highly reproducible chromatin shearing, resulting in improved signal-to-noise ratios and sensitivity for ChIP-seq or ChIP-qPCR.

Pipetting Best Practices for Covaris 96 microTUBE Plate and 8 microTUBE Strip in Automated Liquid Handlers

Application Notes

Covaris labware has multi-well formats, like the 96 microTUBE Plate and 8 microTUBE Strip. The hardware and methods described here maximize the robustness of robot pipetting in Covaris consumables.

Microbiome Sample Preparation: Efficient DNA Extraction and Optional Mechanical Shearing

Application Notes

DNA Extraction

AFA-energetics®-Mediated DNA Extraction from Yeast and Bacteria Using Scanning in a Labcyte 384-well Plate

Application Notes

This document describes the optimized AFA-energetics dose to lyse and extract DNA from S. cerevisiae, E. coli, & L. monocytogenes using a proprietary scanning method 384-well plates.

Nucleic Acid Extraction from Laser-Capture Microdissected (LCM) FFPE Tissues

Application Notes

LCM

Simplifying ChIP from Primary Cells and Clinical Samples for High-throughput Epigenetic Applications

Application Notes

Large-scale analysis of histone marks and transcription factor interactions uses high-quality chromatin, use of validated ChIP-grade antibodies, and optimized reagents to produce meaningful results.

Covaris truSHEAR Mechanical DNA Shearing for NGS Applications

Application Notes

Mechanical DNA Shearing

AFA-energetics®- Mediated RNA Extraction from Yeast

Application Notes

Mediated RNA Extraction from Yeast

Shallow Whole Genome Sequencing Workflow – sWGS

Application Notes

sWGS

Covaris cryoPREP® with AFA: Enabling High-resolution Diagnostic Metagenomics

Application Notes

cryoPREP Applications

Assessing FFPE DNA Quality: The “Illumina FFPE QC Kit” Enables Quantitation of Improvements in FFPE DNA Extraction Technologies

Application Notes

We used the Illumina FFPE QC kit (2) to assess the quality of DNA extracted from two different extraction technologies using four different types of FFPE samples.

AFA-based Sputum Liquification to Enable Downstream Microbial Analysis

Application Notes

Sputim Liquification

Magnetic Bead-based Clean-up Using AFA-energetics®

Application Notes

When precisely tuned, this stream of Covaris Adaptive Focused Acoustic energy enables DNA shearing precisely and reliably to desired fragment sizes, in addition to gentle contact-free sample mixing.

Preparation of whole body Caenorhabditis elegans extracts for chromatin immunoprecipitation using the Covaris® S220 Focused-ultrasonicator

Application Notes

This application note provides a sample preparation protocol using the Covaris S220 that can be used by investigators looking to process whole body C. elegans extracts for ChIP-based applications.

Chromatin Isolation by RNA Purification - ChIRP

Application Notes

ChIRP

Automated NGS Library Construction with Eppendorf epMotion® 5075t/TMX and Covaris® Focused-ultrasonicators

Application Notes

This guide provides users with a complete set of protocols that encompass all Covaris AFA® instruments for DNA shearing to be readily compatible with the Agilent SureSelect Target Enrichment System.

Cross-linked Yeast Chromatin shearing on the Covaris E210

Application Notes

Shearing cross-linked yeast samples on the Covaris E210.

Automated NGS Library Preparation with Beckman Biomek FXP and Covaris E220

Application Notes

Key parameters for high quality library preparation are homogeneity of the library insert size and un-biased fragmentation profile, which is why Covaris AFA technology was chosen to fragment the DNA.

AFA-based MALDI Biotyper Sample Preparation for Mycobacteria Colonies

Application Notes

AFA-based MALDI

Evaluation of Next Generation Library Preparation using DNA Extracted from Dried Blood Spots using truXTRAC® DBS DNA kit

Application Notes

The truXTRAC DBS DNA kit efficiently extracts NGS-grade DNA. AFA enables rehydration and provides cell lysis and mechanical DNA shearing, resulting in NGS library preparation ready DNA.

Cross-linked Yeast Chromatin Shearing on the Covaris Focused-ultrasonicator

Application Notes

The Broad Institute developed a cross-linked chromatin shearing protocol for yeast samples that utilizes the Covaris E210 Focused-ultrasonicator, to be used prior to immunoprecipitation.

HiC Chromatin Immunoprecipitation - Hi-ChIP

Application Notes

Hi-ChIP

Affinity-based Enrichment Analyses of DNA Methylation MCIp, MeDIP, hMeDIP

Application Notes

MeDIP

truCOLLECT^® - Blood Application Note

Application Notes

Transitioning from custom amplicon-based parallel sequencing to hybrid capture-based sequencing in molecular pathology routine diagnostics

Application Notes

Molecular Pahology

High-resolution Chromosome Conformation Capture: Hi-C

Application Notes

Hi-C

Shearing DNA with Confidence: A Robust Method for Generating 3 kb DNA

Application Notes

An Effect of a Shearing Process on the Re-Sequencing of the Arabidopsis thaliana Genome

Application Notes

Shearing Process of Arabedopis

AFA-based Efficient DNA Extraction and Optional Mechanical Shearing from Mycobacteria smegmatis

Application Notes

DNA Extraction from mycobacteria smegmatis.

Cytosine Conversion-based, Genome-wide and Targeted DNA Methylation Analysis

Application Notes

Targeted DNA Methylation Analysis

AFA-based RNA Extraction from Mycobacteria smegmatis

Application Notes

RNA Extraction

Single Cell/Nuclei Isolation from Fresh Frozen Tissue

Application Notes

AFA-based MALDI Biotyper™ Sample Prep for MGIT™ Positive Mycobacteria Cultures

Application Notes

Positive Mycobacteria Cultures

Chromatin Interaction Analysis by Paired-End Tag Sequencing - ChIA-PET

Application Notes

ChIA-PET

Chromosome Conformation Capture Using Biotinylated Oligos - Capture-C

Application Notes

Capture-C

Formaldehyde-Assisted Isolation of Regulatory Elements - FAIRE

Application Notes

FAIRE

Mechanical Fragmentation of RNA using the Covaris Adaptive Focused Acoustics® (AFA®) Technology for Development of mRNA-based Vaccine and Gene Therapy Platforms

Application Notes

Mechanical Fragmentation of RNA using the Covaris Adaptive Focused Acoustics® (AFA®) Technology for Development of mRNA-based Vaccine and Gene Therapy Platforms

truPREP™: Importance of Ligation in Biotechnology and Molecular Biology

Application Notes

Effective disruption of Pseudomonas aeruginosa biofilms using Adaptive Focused Acoustics® (AFA®)

Application Notes

Measuring Sodium Dodecyl Sulfate Carryover In Protein Hydrolysates Prepared via Protein Aggregation Capture

Application Notes

The Covaris PAC workflow is a method for extracting and purifying nucleic acids and liquid chromatography mass spectrometry (LC-MS) ready peptide digests from cultured cells and human tissue. The PAC process was originally designed to function as a protein isolation and purification technique that could be performed in a single vessel. The workflow utilizes paramagnetic beads to indiscriminately bind proteins and to enable the removal of harsh chemicals which are often essential for lysis and solubilization. PAC is highly compatible with a wide range of sample inputs as well as detergents, chaotropes, salts, and solvents [3]. Proteolytic cleavage is performed on-bead, and allows for highly efficient and unbiased sample recovery. The Covaris PAC workflow requires minimal liquid transfer; the workflow starting from cell culture and deparaffinized FFPE tissue can be performed in a single plate format and is fully automatable. The extraction process is facilitated through the use of lysis buffers which contain chaotropic agents or detergents like sodium dodecyl sulfate. While PAC is reputed to generate samples with a high degree of purity, carryover of SDS into the final peptide lysate remains a possible concern due to its suppressive effects on analyte ion signal and fouling of equipment during LC-MS [4]. A method was published describing the colorimetric quantitation of SDS in biological samples [5]. The reported approach relies on measuring the interaction of SDS with the extraction efficiency of methylene blue into chloroform. This application note describes the quantitation of SDS carryover in final tryptic digests of the Covaris PAC workflow to determine if they contain any significant degree of contaminating detergent.

High-Throughput, Low Volume gDNA Extraction from Whole Blood Enabled by Covaris Adaptive Focused Acoustics® (AFA®) and AFA-TUBE™ TPX

Application Notes