PacBio HiFi Sequencing of HG00733

PacBio HiFi data were generated from the HG00733 lymphoblastoid cell line as previously described (G. A. Logsdon et al., 2021 (link)) with modifications. Briefly, DNA was extracted from 4.3×10^6 cells using the Monarch HMW DNA Extraction Kit for Cells and Blood (New England Biolabs) with 1400 rpm lysis speed. After UV absorption and fluorometric quantification (Qubit High Sensitivity DNA kit, Thermo Fisher) on the DS-11 FX instrument (Denovix) and evaluation of DNA integrity on FEMTO Pulse (Agilent), 12 μg of DNA was prepared for sequencing using Megaruptor 3 shearing (Diagenode, settings 19/31) and the Express Template Prep Kit v2 and SMRTbell Cleanup Kit v2 (PacBio). The library was size-selected on a PippinHT instrument (Sage Science) using a 15 kbp high-pass cut. Five SMRT Cell 8Ms were run on a Sequel II instrument using Sequel II chemistry C2.0/P2.2 with 30-hour movie times, 2-hour pre-extension, and adaptive loading targets of 0.8–0.85 (PacBio). Circular consensus calling was performed with CCS version 6.0.0 (SMRT Link v.10.1) and reads with estimated quality scores ≥Q20 were selected for downstream analysis.

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Harvey W.T., Ebert P., Ebler J., Audano P.A., Munson K.M., Hoekzema K., Porubsky D., Beck C.R., Marschall T., Garimella K, & Eichler E.E. (2023). Whole-genome long-read sequencing downsampling and its effect on variant calling precision and recall. bioRxiv.

Publication Preprint 2023

Monarch HMW DNA Extraction Kit for Cells and Blood Qubit High Sensitivity DNA kit DS-11 FX instrument FEMTO Pulse Megaruptor 3 shearing Express Template Prep Kit v2 and SMRTbell Cleanup Kit v2 PippinHT instrument Sequel II chemistry C2.0/P2.2

Adaptive Blood Cell Cell line Fluorometric Library Pulse Sensitivity Smrt

Corresponding Organization : Howard Hughes Medical Institute

Other organizations : Heinrich Heine University Düsseldorf, Jackson Laboratory, UConn Health, Broad Institute

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Variable analysis

independent variables

Megaruptor 3 shearing settings (19/31)
Size selection on PippinHT instrument using 15 kbp high-pass cut
SMRT Cell 8M run on Sequel II instrument using Sequel II chemistry C2.0/P2.2 with 30-hour movie times, 2-hour pre-extension, and adaptive loading targets of 0.8–0.85

dependent variables

Circular consensus reads with estimated quality scores ≥Q20

control variables

DNA extraction from 4.3×10^6 cells using Monarch HMW DNA Extraction Kit for Cells and Blood with 1400 rpm lysis speed
DNA quantification using UV absorption and fluorometric quantification (Qubit High Sensitivity DNA kit) on DS-11 FX instrument
DNA integrity evaluation on FEMTO Pulse
12 μg of DNA used for sequencing library preparation
Express Template Prep Kit v2 and SMRTbell Cleanup Kit v2 used for library preparation

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