SageHLS HMW Library System
Extract. React. Select.
Extract Ultra-HMW DNA from cells
The new SageHLS™ HMW DNA Library System provides a revolutionary approach to DNA extraction whilst preserving chromosomal length DNA (up to 2Mb). This is achieved by performing the extraction procedure in situ of the pre-cast, agarose cassette, thus circumventing excessive shearing of DNA into smaller fragments through the deleterious consequences of excessive pipetting of naked DNA.
Extract directly from cells
- Users load cell suspensions directly into agarose cassettes where a SDS based lysis of cells is performed under electrophoretic conditions.
- Chromosomal length DNA (<2Mb) is immobilised on the inner wall of the agarose cassette, allowing cellular debris and solubilised proteins to migrate through the gel and is removed.
- A cleavase is then added to fragment DNA into electrophoretically-mobile sizes
- Automated DNA size selection collects the DNA in 6 contiguous fragment sizes
- Optical mapping
- Long read sequencing
- Bar-coded linked reads
The SageHLS instrument includes a monitor, keyboard, and mouse. A CPU is built in; an external PC is not required
|Sage HLS specifications|
|Electrophoresis voltage||25V - 100V pulsed field and 150V constant|
|Sample Heating Range||15-60 oC|
|Power requirements||100-240 VAC, 2.5 A, 50-60 Hz|
|Weight||20 lbs / 9 kg|
|Dimensions||12h x 13w x 10d (in) | 31h x 33w x 25d (cm), w/ closed lid|
Kits include 4 or 10 ea. disposable pre-cast gel cassettes. Cassette accommodate 2 samples. Some reagents must purchased separately
HLS-CATCH: Cas-9 Assisted Gene Purification
With customized Cas9 nucleases, isolate large genomic DNA targets with the SageHLS platform. In this example ~200kb targets with collected from NA12878 using a single multiplex SageHLS run.
Eluted fractions with target fragments were identified by qPCR. Pooled target fractions from one SageHLS run (~200-400k target fragments, 10 ng total DNA) were used for library generation using the Agilent SureSelectXT Low Input Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library kit (using manufacturer protocol vs B0, using 11 cycles of pre-enrichment PCR) — without SureSelectXT enrichment and secondary amplification steps.
The library were sequenced on one lane of a HiSeq2500 in Rapid Run mode. Output was aligned to hg38 with BWA-MEM, and PCR duplicates were removed using Samtools. Coverage data was calculated using Bedtools, and displayed in IGV. After removal of duplicates, the Long panel sample produced 186m reads (unique, mapped, pe).
MAPT, Chr 17, 191 kb
Here’s how it works:
- Intact cells or nuclei are loaded into SageHLS cassettes
- Electrophoretic extraction leaves chromosomal-length genomic DNA immobilized in the sample well wall
- Custom Cas9 cleavases perform enzymatic DNA processing
- Size selection electrophoresis moves Cas9-cleaved target regions into the gel channel, away from uncut untargeted genomic DNA in the sample well
- Products are localized by qPCR after electroelution from the gel channel
View the HLS-CATCH workflow schematic
Melissa Smith, Ethan Ellis, James Powell, Ayesha Rasool, Maya Stahl, Robert Sebra
Icahn Institute and Dept. of Genetics & Genomic Sciences
Icahn School of Medicine at Mount Sinai
HLS Software v2.03, Cassette Definition Set 10
SageHLS stage and workflow renaming, March 2018
HLS-CATCH Training Presentation
SageHLS Software Update Instructions
Workflow Guides - Cell Suspension Kits
E.coli Spheroplast Suspension Kit Rev D
Mammalian White Blood Cell Suspension Kit Rev D
Mammalian Tissue Culture Cell Suspension Kit Rev E
SageHLS Operations Manual Rev H
SageHLS Installation Guide Rev A
SageHLS IQ/OQ Guide Rev A
MSDS - Safety Data Sheets
MSDS for HIT0004 and HIT0012 cassette kits
MSDS for HEX0004 and HEX0012 cassette kits
MSDS for CELMWB1 cell extraction kit
MSDS for CELECO1 cell extraction kit
Workflow Guides - Cassette Kits
Lysis Reagent Guide for HEX and HIT kits
HMW DNA Extraction (HEX) Kit Rev F
HLS-CATCH (HIT) kit Rev H
Mammalian White Blood Cells from Whole Blood