Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Summary

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Abstract

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

Introduction

Herbarium collections are a potentially valuable source of both species and genomic diversity for studies including phylogenetics^1,2,3, population genetics^4,5, conservation biology⁶, invasive species biology⁷, and trait evolution⁸. The ability to obtain a rich diversity of species, populations, geographical locations, and time points highlights the "treasure chest"⁹ that is the herbarium. Historically, the degraded ....

Protocol

1. Prior to Start

1. Make fresh cetyl trimethylammonium bromide (CTAB) buffer¹⁷ by adding 20 g of CTAB, 10 g of polyvinylpyrrolidone (PVP) 40, 100.0 mL 1 M Tris pH 8.0, 40 mL of 0.5 M ethylenediaminetetraacetic acid (EDTA) pH 8.0, 280.0 mL of 5 M NaCl, and 400.0 mL of reagent water together, and bring the total volume to 1 L using reagent-grade water. Adjust the pH to 8.0.
   NOTE: Additional reagents can be added to CTAB depending on secondary compounds in individual taxa. See Allen et al.¹⁸ for a thorough list of additive reagents.
2. Add 10 µL of β-mercaptoethanol per 5 m....

Results

DNA Isolation and Final Library Yield
In this study, the efficacy of the protocol for the isolation of herbarium DNA and the recovery of high quality sequencing libraries was demonstrated using fifty different samples with the oldest from 1920 and the youngest from 2012 (Table 2). For each sample, approximately 10 mg of leaf tissue was used for DNA isolation. Greener leaf tissue was favored if available, and no tissue with obvious fungal contamination.......

Discussion

The protocol presented here is a comprehensive and robust method for DNA isolation and sequencing library preparation from dried plant specimens. The consistency of the method and minimal need to alter it based on specimen quality make it scalable for large herbarium-based sequencing projects. The inclusion of an optional reamplification step for low yield libraries allows the inclusion of low quality, low quantity, rare, or historically important samples that would otherwise not be suitable for sequencing.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Veriti Thermal Cycler	Applied Biosystems	4452300	96 well
Gel Imaging System	Azure Biosystems	c300
Microfuge 20 Series	Beckman Coulter	B30137
Digital Dry Bath	Benchmark Scientific	BSH1001
Electrophoresis System	EasyCast	B2
PURELAB flex 2 (Ultra pure water)	ELGA	89204-092
DNA LoBind Tube	Eppendorf	30108078	2 ml
Mini centrifuge	Fisher Scientific	12-006-901
Vortex-Genie 2	Fisher Scientific	12-812
Mortar	Fisher Scientific	S02591	porcelain
Pestle	fisher Scientific	S02595	porcelain
Centrifuge tubes	fisher Scientific	21-403-161
Microwave	Kenmore	405.7309231
Qubit Assay Tubes	Invitrogen	Q32856
0.2 ml Strip tube and Cap for PCR	VWR	20170-004
Qubit 2.0 Fluorometer	Invitrogen	Q32866
Balance	Mettler Toledo	PM2000
Liquid Nitrogen Short-term Storage	Nalgene	F9401
Magnetic-Ring Stand	ThermoFisher Scientific	AM10050	96 well
Water Bath	VWR	89032-210
Hot Plate Stirrers	VWR	97042-754
Liquid Nitrogen	Airgas	UN1977
1 X TE Buffer	Ambion	AM9849	pH 8.0
CTAB	AMRESCO	0833-500G
2-MERCAPTOETHANOL	AMRESCO	0482-200ML
Ribonuclease A	AMRESCO	E866-5ML	10 mg/ml solution
Agencourt AMPure XP	Beckman Coulter	A63882
Sodium Chloride	bio WORLD	705744
Isopropyl Alcohol	bio WORLD	40970004-1
Nuclease Free water	bio WORLD	42300012-2
Isoamyl Alcohol	Fisher Scientific	A393-500
Sodium Acetate Trihydrate	Fisher Scientific	s608-500
LE Agarose	GeneMate	E-3120-500
100bp PLUS DNA Ladder	Gold Biotechnology	D003-500
EDTA, Disodium Salt	IBI Scientific	IB70182
Qubit dsDNA HS Assay Kit	Life Technologies	Q32854
TRIS	MP Biomedicals	103133	ultra pure
Gel Loading Dye Purple (6 X)	New England BioLabs	B7024S
NEBNext dsDNA Fragmentase	New England BioLabs	M0348L
NEBNext Ultra II DNA Library Prep Kit for Illumina	New England BioLabs	E7645L
NEBNext Multiplex Oligos for Illumina	New England BioLabs	E7600S	Dual Index Primers Set 1
NEBNext Q5 Hot Start HiFi PCR Master Mix	New England BioLabs	M0543L
Mag-Bind RXNPure Plus	Omega bio-tek	M1386-02
GelRed 10000 X	Pheonix Research	41003-1
Phenol solution	SIGMA Life Science	P4557-400ml
PVP40	SIGMA-Aldrich	PVP40-50G
Chloroform	VWR	EM8.22265.2500
Ethanol	Koptec	V1016	200 Proof
Silica sand	VWR	14808-60-7
Reamplification primers	Integrated DNA Technologies	see text
Sequencher v.5.0.1	GeneCodes