NDLI: A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants

Content Provider	Springer Nature : BioMed Central
Author	Nishii, Kanae Möller, Michael Foster, Robert G. Forrest, Laura L. Kelso, Nathan Barber, Sadie Howard, Caroline Hart, Michelle L.
Abstract	Background PacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing. Results We developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively. Conclusions DNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.
Related Links	https://plantmethods.biomedcentral.com/counter/pdf/10.1186/s13007-023-01009-x.pdf
Ending Page	11
Page Count	11
Starting Page	1
File Format	HTM / HTML
ISSN	17464811
DOI	10.1186/s13007-023-01009-x
Journal	Plant Methods
Issue Number	1
Volume Number	19
Language	English
Publisher	BioMed Central
Publisher Date	2023-04-29
Access Restriction	Open
Subject Keyword	Plant Sciences Biological Techniques Streptocarpus DNA extraction Genome sequencing Gesneriaceae Long-read sequencing Next-generation sequencing PacBio HiFi SMRTbell™ library
Content Type	Text
Resource Type	Article
Subject	Plant Science Biotechnology Genetics
Journal Impact Factor	4.7/2023
5-Year Journal Impact Factor	5.6/2023

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Low-Input High-Molecular-Weight DNA Extraction for Long-Read Sequencing From Plants of Diverse Families

SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species

Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing

An optimized method for high quality DNA extraction from microalga Prototheca wickerhamii for genome sequencing

A high-quality, long-read genome assembly of the whitelined sphinx moth (Lepidoptera: Sphingidae: Hyles lineata) (Dataset)

Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing.

High molecular weight DNA extraction methods lead to high quality filamentous ascomycete fungal genome assemblies using Oxford Nanopore sequencing.

A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants

Similar Documents

Low-Input High-Molecular-Weight DNA Extraction for Long-Read Sequencing From Plants of Diverse Families

SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species

Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing

An optimized method for high quality DNA extraction from microalga Prototheca wickerhamii for genome sequencing

A high-quality, long-read genome assembly of the whitelined sphinx moth (Lepidoptera: Sphingidae: Hyles lineata) (Dataset)

Optimization of long-range PCR protocol to prepare filaggrin exon 3 libraries for PacBio long-read sequencing.

High molecular weight DNA extraction methods lead to high quality filamentous ascomycete fungal genome assemblies using Oxford Nanopore sequencing.

A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants