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Recently Published Protocols

Using automated size selection tools, researchers are continually finding new ways to improve sequencing methods. The following protocols have been published in the past year.

 

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EpiRAD-seq: Methods in Ecology and Evolution, August, 2015
EpiRADseq: scalable analysis of genome-wide patterns of methylation using next-generation sequencing
A reduced representation approach to methylation assessment from scientists at the University of Texas. This uses epigenetic modification of the ddRAD-seq application (another technique enabled by Pippin; see Peterson et al.) using a methylation-sensitive restriction enzyme.

Mate Pair library construction: Biotechniques, July 2015
A method to simultaneously construct up to 12 differently sized Illumina Nextera long mate pair libraries with reduced DNA input, time, and cost
In a powerful application of the SageELF fractionator from The Genome Analysis Centre (TGAC), 12 mate pair libraries are prepared from 10ug of DNA to cover a wide range of jump sizes.

ddRAD-seq for Ion Proton: Molecular Ecology Resources, April 2015
Double-digest RAD sequencing using Ion Proton semiconductor platform (ddRADseq-ion) with nonmodel organisms
An Ion Proton ddRAD-seq protocol is described here and compared to the Illumina-based method from Peterson et al..

Reduced Representation Bisulfite Sequencing: JoVE video, February 2015
Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
In this video protocol, researchers from Weill Cornell and University of Michigan use Pippin Prep to provide highly accurate Illumina library size selection to improve the resolution of RRBS.

Paired-End library construction for 454: Nature Protocol Exchange, August 2014
Modified paired end rapid library preparation protocol for 454 GS Junior 8 kb library preparation using Covaris g-tubes and BluePippin electrophoresis
The BluePippin is used to size select 8kb fragments for this improved protocol from scientists at the University of Saskatoon.

 

 

Validated Supplier Protocols
Automated size selection improves many library workflows, particularly when working with sheared genomic DNA. Protocols that recommend gel purification, as well as clean up steps, are often enhanced by automated DNA sizing. Sage Science instruments have been written into a number of supplier protocols when automated size selection is particularly useful.

 

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Illumina:
Nextera Mate Pair Sample Preparation Guide

TruSeq Synthetic Long-Read DNA Library Prep Guide

Ion Torrent (login to Ion Community to access):
Ion Xpress™ Plus gDNA Fragment Library Preparation
Ion Xpress™ Plus and Ion Plus Library Preparation for the AB Library Builder System

PacBio:
Third-generation long-read technologies such as PacBio RSII single molecule sequencing derive maximum performance when presented with high molecular weight DNA libraries. Automated size selection has been particularly useful for increasing average read lengths. This reference chart summarizes the PacBio applications and protocols that benefit from Sage products.

RNA protocols:
New! Bioo Scientific: Size Selection of NEXTflex™ Small RNA Sequencing Kit v2 Libraries Using the Sage Pippin Prep System

New England Biolabs: Automated Size Selection of NEBNext Small RNA Libraries with the Sage Pippin Prep

 

 

More Resources
Can your application benefit from size selection? These publications cite DNA size selection steps and provide details on how the Sage platform is used.

 

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Paired-end sequencing: Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer
Illumina paired-end library construction is the most popular use of the Pippin platform, and this is a typical citation. We are proud to have our products cited in the context of great research.

Mate-pair sequencing: Optimization and cost-saving in tagmentation-based mate-pair library preparation and sequencing
This paper suggests some cost-saving and optimization tips for using the Illumina Nextera Mate Pair kit.

Long-read sequencing: Reconstructing complex regions of genomes using long-read sequencing technologyThis groundbreaking paper from the Eichler lab at the University of Washington highlights the power of the PacBio platform, and cites the BluePippin for SMRTbell library prep.

MicroRNA sequencing: Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing
This describes three sample prep workflows: manual gel purification, AMPure beads, and automated size selection with the Pippin Prep.

ChIP-seq: Genome-wide comparison of PU.1 and Spi-B binding sites in a mouse B lymphoma cell line
Automated size selection is commonly used for ChIP studies. In this paper, transcription factor associated DNA is sequenced.

HLA typing: Towards allele-level human leucocyte antigens genotyping – assessing two next-generation sequencing platforms: Ion Torrent Personal Genome Machine and Illumina MiSeq
The Omixon Holotype HLA kit requires a Pippin Prep and Illumina MiSeq and uses methods developed at the Children’s Hospital of Pennsylvania. In this publication, researchers also suggest methods for Ion PGM users.

ddRAD-seq: Assessing the utility of whole genome amplified DNA for next-generation molecular ecology
The authors show comparability between ddRAD-seq results from low amounts of DNA subject to whole genome amplification (WGA) and ddRAD-seq results from raw genomic DNA.

RAD GBS: Genotype-by-sequencing of the plant-pathogenic fungi Pyrenophora teres and Sphaerulina musiva utilizing Ion Torrent sequence technology
This paper provides a comprehensive method for genotyping-by-sequencing using Pippin Prep and the Ion PGM.

MeDIP-Seq: Arterial endothelial methylome: differential DNA methylation in athero-susceptible disturbed flow regions in vivo The researchers used Pippin size selection and Diagenode’s MagMeDIP kit for this differential methylation study.

More publications that cite Sage products can be found here.

 

 

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