Recently Published Protocols

Using automated size selection tools, researchers are continually finding new ways to improve sequencing methods.



Enrichment for microbial DNA in human saliva samples, Microbiome, 2018
Improving saliva shotgun metagenomics by chemical host DNA depletion
Scientists from the University of California, San Diego, developed a rapid, cost-effective protocol for enriching microbial DNA collected from fresh and frozen human saliva samples. They used PippinHT to remove primer dimers and target 300 bp to 800 bp DNA fragments prior to sequencing.

Sample prep for small RNA, Extracellular RNA, 2018
Preparation of Small RNA NGS Libraries from Biofluids
In this book chapter, scientists present an NGS protocol designed for use with low-input biofluid samples containing extracellular RNAs. Modifications reduce sequence-specific bias seen in many commercially available small RNA prep kits. The protocol includes PippinHT, though authors note that Pippin Prep and BluePippin can also be used to reduce variability in gel excision.

Enrichment for circulating tumor studies, bioRxiv preprint, 2017
Selecting Short DNA Fragments In Plasma Improves Detection Of Circulating Tumour DNA
By using size selection to target ctDNA, scientists from Cancer Research UK and Cambridge University Hospitals were able to significantly enrich for the DNA of interest. They used PippinHT to perform the key sizing step.

Targeted capture of large genomic regions, Nature Communications, 2015
Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters
This method uses elements of CRISPR to target large genomic regions — genes or large elements — for NGS workflows. It’s a cost-effective alternative to whole genome sequencing for scientists who need a portion of DNA that’s too big or repetitive to amplify easily. When paired with the SageHLS, the method offers excellent recovery and reproducibility.

EpiRAD-seq: Methods in Ecology and Evolution, 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, 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, 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, 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, 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.



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

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.



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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