Best practices: Sage Science size selection with Diagenode Megaruptor shearing for long-read sequencing library preparation
This is an in-depth collaboration between Sage and Diagenode/Hologic. Megartupter 3 DNA shears of different sizes were subject to size selection as part of our “Range+T” evaluation for PippinHT and BluePippin. Data is provided which provide the best practices for achieving DNA size selections with tight fragment distributions around median sizes from 9-30kb for long-read sequencing applications.
Range + T Size Selection Method
Increases PacBio HiFi Read Lengths
From the Arizona Genomics Institute, University of Arizona College of Arizona College of Agricultural & Life Sciences. Researchers were given early access to the Range + T programming mode. The PippinHT was used for size selection on PacBio SMRTbell library construction for HiFi Sequencing of plant genomes. High Quality sequencing reads were obtained with mean subreads of 20-25kb, indicating that Range + T can increase current HiFI readlength recommendations.
Haplotype-resolved Sequencing the Major Histocompatibility Complex (MHC) Locus using HLS-CATCH™ Target Enrichment and TELL-Seq™ Linked Read Sequencing
From Universal Sequencing and Sage Science, HLS-CATCH and UST TELL-Seq linked reads are used to Haplotype-resolve the entire MHC complex. The MHC locus was purified as 20X 400kb fragments using the CATCH process.
HMW DNA Size Selection with the HLS2 instrument and a >50kb High-Pass Protocol
The method for DNA size selection for fragment ranges starting around 50 kb and up to 2MB is described
Higher Accuracy “Range + T” Size Selection: BluePippin and PippinHT
A method for higher accuracy size selection on the BluePippin and PippinHT is outlined. “End” values may be entered in timed mode to account for the fragment size distribution of the input DNA sample.
Targeted Mitochondrial DNA Extraction and Enrichment Using the SageHLS System
A novel approach for enriching mitochondrial DNA for sequence analysis without requiring PCR or gradient centrifugation.
Accurate Cost-effective Haplotype-resolved Sequencing of Large Targeted Genomic Regions Using HLS-CATCH™ Sample Prep with TELL-Seq™ Library Preparation
From Universal Sequencing and Sage Science, HLS-CATCH and UST TELL-Seq linked reads are used to Diplotype full-length BRCA2 targets from a model Ashkenazi Trio
PippinHT: Collection of <167 bp DNA for Cell-Free DNA Library Construction
Parameters for timed DNA size selection for collection of fragments below 167bp are provided for the PippinHT platform. Studies have shown that shorter cfDNA fragments (40 -167bp) retain genetic information about the fetus,
tumor associated copy number aberrations or the cellular origin of cfDNA,
DNA Size Selection for PacBio® Hi-Fi™ CCS Reads: New SageELF™ Waveforms
SageELF timed protocols are presented that can be used to collect narrow fragment size distributions of sheared DNA for PacBio’s Hi-Fi library prep.
A Simple Screening Assay for
C9orf72 ALS Repeat Expansions
A collaboration with the New York Genome Center to propose a method for determining the number of C9orf72 repeat expansions using a restriction digest approach with the SageHLS platform.
Targeted Mitochondrial DNA Extraction and Enrichment Using the SageHLS System
A novel approach for enriching mitochondrial DNA for sequence analysis without requiring PCR or gradient centrifugation.
Illumina Sequencing of Gene
Targets with HLS-CATCH™
In a collaboration between Icahn School of Medicine at Mt. Sinai, the principle of HLS-CATCH is demonstrated. Full-length targets of BRCA1, MAPT, and MUC13 are purified and sequenced using the Illumina platform
Size Selection of Long-chain Fragments (8kb)in Nextera Mate Pair Library Preparation
From Sage’s Japanese partners, Nippon Genetics and the University of Miyazaki School of Medicine. Recommendations for DNA size selection with the Illumina Mate Pair library construction kits.
ChIP-Seq Library Prep
A 2011 case study with the Dana Farber Cancer Institute using the Pippin Prep for ChIP-Seq.
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