Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Illumina

sequencing

and

assessment

of

new

cost-efﬁcient

protocol

for

metagenomic-DNA

extraction

from

environmental

water

samples

Mariam

Hassan

a,∗

_,

_Tamer

_Essam

a

_,

_Salwa

_Megahed

a,b

a_Cairo_University,_Faculty_of_Pharmacy,_Department_of_Microbiology_and_Immunology,_Cairo,_Egypt

b_October_University_for_Modern_Sciences_and_Arts_(MSA),_Faculty_of_Pharmacy,_Department_of_Microbiology_and_Immunology,_Cairo,

Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21October2017

Accepted14March2018

Availableonline3April2018

AssociateEditor:NiltonLincopan

Keywords: Bioinformatics Method Microbiome Next-generationsequencing QIIME

a

b

s

t

r

a

c

t

Inthisstudy,thedevelopmentandassessmentofamodified,efficient,andcost-efficient

protocolformDNA(metagenomicDNA)extractionfromcontaminatedwatersampleswas

attempted. Theefﬁciency ofthedeveloped protocolwasinvestigatedincomparison to

awell-establishedcommercialkit(Epicentre,MetagenomicDNAIsolationKitforWater).

Thecomparisonwasintermsofdegreeofshearing,yield,purity,duration,suitabilityfor

polymerasechainreactionandnext-generationsequencinginadditiontothequalityof

next-generationsequencingdata.The DNAyieldobtained fromthedeveloped protocol

was2.6foldshigherthanthatofthecommercialkit.Nosigniﬁcantdifferenceinthealpha

(Observedspecies,Chao1,SimpsonandPDwholetree)andbetadiversitywasfoundbetween

theDNAsamplesextractedbythecommercialkitandthedevelopedprotocol.Thenumber

ofhigh-qualitysequencesofthesamplesextractedbythedevelopedmethodwas20%higher

thanthoseobtainedbythesamplesprocessedbythekit.Thedevelopedeconomicprotocol

successfullyyieldedhigh-qualitypuremDNAcompatiblewithcomplexmolecular

applica-tions.Thusweproposethedevelopedprotocolasagoldstandardforfuturemetagenomic

studiesinvestigatingalargenumberofsamples.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Abbreviations:NGS,next-generationsequencing;PCR,polymerasechainreaction;mDNA,metagenomicDNA.

∗ _{Corresponding}_author_at:_Faculty_of_Pharmacy_Cairo_University,_Kasr_El-Aini_Street,₁₁₅₆₂_Cairo,_Egypt.

E-mail:maryam.hiekal@pharma.cu.edu.eg(M.Hassan).

https://doi.org/10.1016/j.bjm.2018.03.002

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Introduction

Recently,theevolutionofthe disciplineof“Metagenomics”

hasgraspedconsiderableattentionworldwidewith

tremen-dousexpectations.1_Several_ecosystems_are_still_untapped_and

willbestudiedandexploredthroughmetagenomicstudies.

Thishasbeenfacilitatedbythevastadvancesinmolecular

toolssuchasthenext-generationsequencing(NGS).2,3

However, an important prerequisite for a successful

metagenomic analysis is the use of an efﬁcient protocol

forextracting whole community DNA from environmental

samples.4 _Therefore, _a _mDNA _(metagenomic _DNA)

extrac-tion protocolhasbeen considered tobethemilestone and

themaingatetoaccurate,reliable, andsuccessful

metage-nomic analyses.5 _Accordingly,_the _isolation _of_high-quality

DNAthatcoversthewholemicrobialdiversityintheoriginal

samplewas phrasedasalimiting stepfortheconstruction

ofmetagenomiclibrariesandotherDNAbasedmetagenomic

projects.6

Intheseregards,manystudies havereportedthe

devel-opment of methods and even commercial kits for mDNA

extraction.5,6 _However,_still, _there_is _a_surge _in_developing,

improving,andoptimizingneworcurrentprotocolsformDNA

extractionfromdifferenttypesofsamples.Ametagenomic

DNAextractionmustbefollowedbyaqualitycontrolofthe

extractedDNA.Despitetherecentadvancesinthe

molecu-lartools,many researchersstilluserelatively conventional

moleculartechniquessuchasPCR,restrictiondigestion,and

cloningfortestingthequalityoftheextractedmDNA.7–9_In

thisperspective,thecurrentstudydevelopedamodiﬁed

pro-tocolfortheextractionofmDNA fromcontaminatedwater

sampleswiththeintroductionofNGSasaqualitycontroltool

tomeasuretheefﬁciencyoftheextractionprotocolandthe

qualityoftheextractedDNA.ThequalityoftheextractedDNA

wascomparedtothatofawellestablishedcommercialkitin

termsofdegreeofshearing,yield,purity,timerequirement,

suitabilityforPCRandNGS,andthequalityofNGSdata.

Materials

and

methods

SamplescollectionsandpreparationforDNAextraction

Industrial wastewater from a coal coking factory in Egypt

(sampleA)wasusedinthisstudy.Thesampleswerecollected

from the biologicalwastewatertreatment plantatthe

fac-tory.Otherwatersamples(13samples,namedasB–N)were

alsocollectedfromaphoto-bioreactorusinganalgal-bacterial

systemtotreatcokingwastewater.

The sufﬁcient volume of the samples was calculated

accordingtoapredeterminedrelationshipbetweenthedry

weightofbiomassand thesamplevolumetoreach aﬁnal

biomassdryweightof1.7mgfortheDNAextraction.These

volumeswere immediately ﬁlteredusingasterileﬁltration

unit (Glassco®, India) and vacuum pump (Pro-set, CPS®,

Germany). The samples were ﬁltered using sterile 0.2␮m

poresizecellulosenitratemembraneﬁlters(47mmdiameter,

Sartorius®,Germany).Themembraneﬁlterswerethenstored

frozenat–20◦CtillDNAextraction.

DNAextractionusingcommercialkit

Metagenomic DNA isolation kit for water (Epicentre®, USA)

wasusedaccordingtothemanufacturer’sinstructions.The

extractedDNApelletswerere-suspendedinmoleculargrade

water(DNaseandRNasefreewater)andstoredat−20◦_C_till

furtheruse.

DNAextractionbythedevelopedapproach(chemical protocol)

Severaltrialswereattemptedtooptimizeaprotocolforthe

extractionofenvironmentalmDNA.Theoptimizedprotocol

was achieved throughthe followingsteps: a 0.2-␮m

mem-braneﬁltercarryingthemDNAwasasepticallyplacedatthe

centerofasterile15-mLfalcontube.Then5mLofextraction

buffer(1%,w/vcetyltrimethylammoniumbromide(CTAB),3%,

w/vsodiumdodecylsulfate(SDS),100mMTris–HCl,100mM

NaEDTA,1.5MNaCl,pH8.0)wasaddedtothefalcontube.The

resulting solutionwas incubatedin awaterbath(65–70◦C)

for 60min with intermittent vortexing. The content was

centrifuged for15minat4500×g andthe supernatantwas

transferredintoanewsterilefalcontube.Isopropanol(4mL)

was liquated to the supernatant and incubated on icefor

20min.The content was centrifuged at−4◦_C _and ₄₅₀₀_×_g

for15minandthesupernatantwascarefullyaspiratedand

discarded.TheDNApelletswerewashedwith200␮Lof70%

ethanolandcentrifugedat−4◦Cand4500×gfor10min.Again

thesupernatantwasdiscardedbycarefulaspirationandthe

DNApelletswerefully driedinalaminarﬂowcabinet.The

dried pellets were re-suspended in100␮L moleculargrade

water(DNaseandRNasefreewater)andstoredat−20◦_C_till

furtheruse.

FurtherpuriﬁcationoftheextractedmDNA

Thepreviouslymentionedprotocolwasusedforfurther

puriﬁ-cationoftheextractedmDNAusinggelpuriﬁcationtechnique.

Where 25␮L of the extracted mDNA was resolved on an

agarosegelin0.8%TAEbufferedagarosegelandvisualized

usingethidiumbromide.DNAbandwasthensliced

(Supple-mentary Fig. S1-A) and puriﬁedusing UltraClean® 15 DNA

Puriﬁcation Kit (Mo Bio®, USA) according to the

manufac-turer’sinstructions.

DNAdetectionbygelelectrophoresisandnanophotometer

ThesizeanddegreeofshearingoftheextractedDNAwere

val-idatedbycomparisontoafosmidcontrolDNAwithasizeof

40kbandconcentration100ng/␮L(providedinthe

Metage-nomic DNA Isolation Kit forWater, Epicentre). Thefosmid

controlDNAandtheextractedmDNAwereelectrophoresed

in0.8%TAEbufferedagarosegelandwere visualizedusing

ethidiumbromide.

The quantiﬁcation of the extracted mDNA was done

by a spectrophotometric method using nanophotometer

(Implen, NanoPhotometer® P-330, Germany).10 _The _purity

(3)

ratios at 260/230nm and 260/280nm calculated using the

nanophotometer.10–12

PCRampliﬁcation

The quality of the extracted mDNA was also assessed

by its liability to enzymatic reactions using PCR. This

also tested the presence of any impurities that might

inhibit the enzymatic reactions. Two universal 16S

ribo-somal DNA primers 28F 5AGAGTTTGATCCTGGCTCAG-3

(positions 8–28 in Escherichia coli numbering) and 1512R

5ACGGCTACCTTGTTACGACT-3(positions1512–1493inE.coli

numbering)wereused.13 _The_positive_control_employed_the

genomicDNAextractedfrom Pseudomonasaeruginosa(ATCC

9027),whilethenegativecontrolwasdevoidofanyDNA

tem-plate.

DNAsequencing

Illumina-MiSeqTM _sequencing ₍₂_×₃₀₀_bp _paired-end

protocol) was performed using the universal primers

341F 5-CCTACGGGNGGCWGCAG-3 and 805R 5

-GACTACHVGGGTATCTAATCC-3 to target the V3 and V4

regionsofthe16SrRNAgene.14_The_used_sequencing_kit_was

MiSeqReagentKitV3,600cycle(Illumina,USA).

Availabilityofsequencingdata

The 16S sequence data were submitted to the Sequence

Read Archive (SRA) under Bioproject (PRJNA353621) and

SRAstudy(SRP093422).ThedatahavebeenreleasedbyNCBI

(https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP093422)

and assigned the accession numbers: SRR5028842,

SRR5028843, SRR5029151, SRR5029152, SRR5029153,

SRR5029154,SRR5029155,andSRR5029156.

NGSdataanalysis

Illuminasequencingreadswereprocessedusingthesoftware

QIIME“QuantitativeInsightsIntoMicrobialEcology”,version

1.9.1 as described in detail in the supplementary data.15

Allstatisticalanalyses,unlessspeciﬁedotherwise,were

per-formedusingpythonscriptsimplementedinQIIMEversion

1.9.1(asdescribedindetailinthesupplementarydata).

Results

DetectionofextractedmetagenomicDNAandquality assurance

In this study, three sets of metagenomic DNA samples,

referredtoas thekit extracted samples,chemical protocol

extractedsamples,and gel-extractedsampleswere

consid-ered.Thethreeinvestigatedmethodsyieldedmetagenomic

DNAwithasizeintherangeof40kb,comparedtothefosmid

control (Supplementary Fig. S1-B). The extracted

metage-nomicDNAshowedasinglebandwhenresolvedonanagarose

gel (Supplementary Fig. S1) with no other sheared bands.

Thehumiccontaminantswereseenintheagarosegelofthe

metagenomicDNAextractedbyboththeEpicentrekitandthe

chemicalprotocols,yetthishumicsubstancewascompletely

absentinthegelextractedDNAsamples(SupplementaryFig.

S1-B).

ThedevelopedchemicalprotocolrecordedthehighestDNA

yieldof21±5␮g/mgbiomass,whiletheEpicentrekitrecorded

signiﬁcantly lower DNA yield of 8±2␮g/mg biomass (one

wayANOVA,p-value=0.0026) (Fig. 1-A).Onthe otherhand,

themetagenomicDNAextractionbygelpuriﬁcationmethod

showed asigniﬁcantly lowyieldof4±1␮g/mgofbiomass,

when comparedtothe yieldofthe chemical protocol(one

wayANOVA,p-value=0.0026).Yet,there wasno signiﬁcant

difference between the DNA yieldedbyboth the Epicentre

kitandgelpuriﬁcationmethod(t-test,p-value=0.1032).The

same pattern was observed with the DNA concentrations,

wherethechemicalprotocolsigniﬁcantlyrecordedthehighest

DNA concentration of 690±180ng/␮L (one way ANOVA,

p-value=0.0007)(Fig.1-B).WhilethelowestDNAconcentration

(33±9.6ng/␮L)wasrecorded bythegelpuriﬁcationmethod

(Fig.1-B).

Theefﬁciencyofremovingproteincontaminationfromthe

metagenomic DNA wasvalidated bythe absorbance ratios

at 260/280nm and was greater than 1.7for all three used

extractionmethods(Fig.1-C).Therecordedabsorbanceratios

at260/230nm fortheEpicentre kit protocol andthe

devel-opedchemicalprotocolwere1.5±0.1and1±0.1,respectively

(Fig.1-D).However,thegelpuriﬁcationmethodshowed

sig-niﬁcantlylowabsorbanceratiosat260/230nmof0.1±0.2(one

wayANOVA,pvalue<0.0001)(Fig.1-D).

The quality ofthe extracted metagenomic DNA and its

suitability fordownstreamprocessingwere investigatedby

usingPCR.ThemetagenomicDNAextractedbythethree

eval-uated methods(kit, chemical and gel puriﬁcation) showed

successfulPCRampliﬁcationofthetargetedsequencesof16S

ribosomalDNA(SupplementaryFig.S2).

MetagenomicDNAsequencing

In order to test the suitability of the extracted

metage-nomic DNAtomorechallenging and complexdownstream

processingandfurthervalidatethepurityandqualityofthe

extractedDNA,thesamplesweretestedfortheirﬁtnessto

Illu-minaMiSeqTM_{next-generation} _sequencing_(NGS)._Since_the

DNAextractedbygelpuriﬁcationmethodresultedinthe

low-estconcentration(Fig.1-B),thismethodwasexcludedfrom

theNGSanalysis.FourmetagenomicDNAsamples(A,B,C,

and D) processedby each Epicentre kit and the developed

chemicalprotocolwereconsideredinthisinvestigation(atotal

of8samples).

Thenumberofhigh-qualitysequencesobservedwiththe

metagenomic DNAsamplesextracted bythechemical

pro-tocol(101,109±3563) washigherthan thatobservedbythe

Epicentrekit samples(85,026±12,320).However,this

differ-encewasnotsigniﬁcant(t-test,p-value=0.0711).Again,there

wasnosigniﬁcantdifferencebetweenthenumberofobserved

OTUs in the metagenomic DNA samples extracted by the

developedchemicalprotocol(1718±365.1)andthatextracted

bytheEpicentrekit(1796±373.2)(t-test,p-value=0.886).

Atotalof44phylawereidentiﬁedinboththe groupsof

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Kit Chemical Gel 0 20 40 60 80 ug / mg

Yield(ug DNA /mg Biomass)

*

A

* Kit Chemical Gel 0 2 4 6 8 10 12 14 16 18 A 260 /280 A 260 / 280 * *

C

Kit Chemical Gel 0 300 600 900 1200 1500 1800 ng/ul

DNA Conc. (ng/ul)

*

B

*

Kit Chemical Gel 0.0 0.5 1.0 1.5 2.0 2.5 A260 /230 A 260 / 230 * * *

D

Fig.1–ScatterdotplotsofcomparativeanalysisofthemetagenomicDNAextractedusingthethreestudiedmethods(kit, chemicalprotocol,andgelextractionmethod).(1-A)DNAyieldexpressedasmicrogramsofDNApermilligramofbiomass. (1-B)DNAconcentrationexpressedasng/␮L.(1-C)Absorbanceratiosat260/280nm.(1-D)absorbanceratiosat260/230nm. (*)meansthatthereisasigniﬁcantdifferenceatp-value<0.05(One-wayANOVA,Tukey’smultiplecomparisontest).

signiﬁcantdifferenceintherelativeabundanceofeach

phy-lumbetweenthetwogroupsofthesamples(Kruskal–Wallis

testwithgroupsigniﬁcance.py,p-value>0.05)(Fig.2).Thefour

majorbacterialphylaidentiﬁedinthesampleswere

Proteobac-teria(Gram-negative),Firmicutes(Gram-positive),Bacteroidetes

(Gram-negative),andDeferribacteres(Gram-negative)(Fig.2).

Thealphadiversity (withincommunity diversity)inthe

samplesextractedbybothprotocols(kitandchemical

proto-col)wasdeterminedandcompared.Therewasnosigniﬁcant

difference (non-parametric Kruskal–Wallis test with

com-pared alpha diversity.py, p-value=0.682) in the observed

species richness (number of unique OTUs per number of

sequences)betweenthe samplesextracted byEpicentrekit

andthechemicalprotocol(Fig.3-A).Therarefactioncurvesof

thetruerichness(Fig.3-A)werecomparedtothecurvesofthe

estimatedspeciesrichness(indicatedbychao1richness

esti-mator)(Fig.3-B).Therarefactioncurvesshowingtheestimated

richness(Fig.3-B)ofbothprotocols(kitandchemicalprotocol)

weresuperimposedandnosigniﬁcantdifferencewasrecorded

betweenthetestedprotocols(non-parametricKruskal–Wallis

testwithcomparealphadiversity.py,p-value=0.962).

Simpson’s index rarefaction curves for both methods

(kit and chemical protocol) were completely superimposed

(Fig. 3-C), with no signiﬁcant difference (non-parametric

Kruskal–Wallis test with compare alpha diversity.py,

p-value=0.952).Thesamplesextractedbybothmethods(kitand

chemicalprotocol)reachedaplateauafter>16,000sequences

(Fig.3-C).Thisindicatedthatthesequencingdepthwas

suc-cessfulincapturingtheexistingmicrobialdiversityinallthe

investigatedsamples.

The PD whole tree (alpha diversity measure) was used

to represent the phylogenetic diversity between samples

extractedbyEpicentrekit andthe developedchemical

pro-tocol(Fig.3-D).Therewasnosigniﬁcantdifferencebetween

the two investigated protocols in terms of PD whole tree

(non-parametric Kruskal–Wallis test with compared alpha

diversity.py,p-value=0.984).

To investigate the beta diversity (between community

diversity)betweenthemetagenomicDNAsamplesextracted

by the Epicentre kit and developed chemical protocol, the

UniFrac distancemetrics were used,as UniFractakes

phy-logenetic relatedness into account while computing beta

diversity.Toexplorethedifferencesinoverallmicrobial

com-munitycompositionacrossthe metagenomicDNAsamples

extractedbythetwoinvestigatedprotocols,boththe

phyloge-neticunweightedUniFracdistances(Fig.4-A)(consideronly

thepresenceorabsenceoftaxa)andweightedUnifrac(Fig.4

-B)(considertaxonrelativeabundance)werecomputed.The

principal coordinatesanalysisplot(PCoA),showedthat the

metagenomic DNAfrom the samesamplesourceclustered

togetherregardlessoftheusedmethodofextraction(Fig.4).

Similarly,thebetadiversityanalysisofthesamplesshowed

thateach samplesourcerepresentedadistinctmicrobiome

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0ther — Ar matimonadetes Actinobacter ia Acidobacter ia BRC1 _AC 1 WWE1 WS6 WS5 Verr u comicrobia Unass ign ed Ther motog ae Ther mi Tener icutes TP D-58 TM7_TM6 SynergistetesSpirochaetes SR1 SAR406 0% 10% 20% 30% 40% Proteobacteria 60% 70% 80% 90% 100% Planctom ycetes 0P8 0P3 0P11 0P1 0D1 Nitrospir a e NKB19 0p9 0% 10% 20% 30 % 40 % 50 % 60% 70% 80% 90% 100 % Gemmatimonadetes GN02 Fusobacter ia Firm icutes Elusimicrobia EM3 0% 10% 20% 30% 40% Developed._protocol 60% 70% 80% 90% 100% Def erib_acteres Cy anobacter ia Chlorofle xi Chlorobi Chlam ydiae Caldiser ica Bacteroidetes 100% 90% 80% 70% 60% 40% 30% 20% 10% 0% K it — — — — — — — — — — _—— — — — —— — — — — — — — — — — — — — _— — — — —— — — —

Fig.2–Therelativeabundanceoftheidentiﬁedphylainthesamplesextractedbythedevelopedchemicalprotocol

(Chemical)andthoseextractedbythecommercialEpicentrekit(Kit).Intheﬁgureeachphylumisrepresentedbyacircularly arrangedribbon,whosewidthisproportionaltoitsrelativeabundance.

Thesigniﬁcanceofthe usedDNAextractionmethodon

the overall microbial community composition was tested

usingpermutationalmultivariateANOVA.Therewasno

sig-niﬁcanteffectofthemetagenomicDNAextractionmethods

usedintheoverallcommunitycompositionwithinthe

indi-vidualsamples (PERMANOVA, pvalue=0.816 and ANOSIM,

p-value=0.813).

Discussion

Cokingwastewaterisacomplexhighlycontaminated

indus-trialefﬂuent,whichhasbeenstudiedinagreatdetail.16_Hence,

cokingwastewatersamplesfrombothcoalcokingfactoryand

photosyntheticallyaeratedbioreactorwereselectedforthis

study.Thedevelopedchemical protocolforthe isolationof

metagenomicDNAfromthecontaminatedwatersampleswas

comparedtoacommercialkit(MetagenomicDNAisolationkit

forwater,Epicentre,USA),asitisawell-establishedkitthat

hasbeenusedthroughrecentmetagenomicstudies.17

Inthedevelopedprotocol,CTABandSDSwereusedas

puri-fyingagentsfortheremovalofphenoliccontaminantsand

humicsubstancesasdescribedearlierinmanyDNA

extrac-tionprotocols.8_The_inclusion_of_purifying_agents_in_the_DNA

extractionbufferissufﬁcienttoyieldDNAwithanadequate

puritythatcomplieswiththedifferentmolecularapplications.

Thiseliminatestheneedforfurtherstepswithaseparate

puri-fyingbufferoranyadditionalpuriﬁcationstepsasreportedby

otherpreviouslystudiedDNAextractionprotocols.5,9,11

There-fore,thisaddsextrabeneﬁtsofthedevelopedprotocolinterms

oftimeandmoneysaving.

Althoughtheuseoforganicsolvents(phenol/chloroform)

iscommon inthe previouslydevelopedmetagenomic DNA

extractionprotocols,18_yet_the_use_of_such_hazardous_solvents

wasavoidedintheprotocoldevelopedinthisstudy.Several

studieshaveattemptedtoenhancethequalityandyieldof

themetagenomicDNAextractedfromenvironmentalsamples

byusingphysicaltreatments(beadbeatingand sonication)

intheextractionprotocols.However,theseharshtreatments

usuallycause shearing ofDNA,which makesit unsuitable

forfurtherdownstreamprocessing.8_Accordingly,_these_severe

treatmentswerenotincludedinthedevelopedchemical

pro-tocol,yieldingun-shearedmetagenomicDNAwithasizeof

≈40kb.

TheyieldofDNAinthedevelopedchemicalprotocolwas

signiﬁcantlyhigher(2.6folds)thanthatoftheEpicentrekit

(one-wayANOVA,p-value=0.0026).Similarobservationshave

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Observed species: protocol Chaol: protocol

A

C

D

B

3000 4000 3500 3000 2500 2000 1500 1000 500 0 0 2500 2000 1500 1000 500 1.0 150 100 50 0 0 0.8 0.6 0.4 0.2 0.0 0 20000 40000 60000 80000 100000 20000 40000 60000 80000 100000 0 0 20000 40000 60000 80000 100000 20000 40000 60000 80000 100000 Sequences per sample

Sequences per sample Sequences per sample Sequences per sample Simpson: protocol

PD whole tree: protocol Ch Ep Ch Ep Ch Ep Ch Ep Raref action measure: obser v ed_species Raref action measure: PD_whole_tree Raref action measure: simpson Raref action measure: chaol

Fig.3–RarefactioncurvesofalphadiversitymeasuredforthemetagenomicDNAsamplesextractedbythedeveloped chemicalprotocol(Ch)andthecommercialEpicentrekit(Ep).(3-A)Theobservedspeciesindicatingthetruespecies richness,(3-B)Chao1richnessestimatorindicatingestimatedspecies’richness,(3-C)Simpson’sindexindicatingspecies’ evennessand(3-D)PDwholetreeindicatingthephylogeneticdiversity.

A

B

PC2 (24.49 %) PC3 (16.84 %) PC1 (38.04 %) PC3 (18.26 %) PC1 (46.07 %) PC2 (31.46 %) A.kit B.Kit A.ch A.kit A.ch C.kit C.ch B.Ch C.Kit C.Ch D.Kit D.Ch B.Kit B.Ch D.KitD.Ch Ch Ep Ch Ep

Fig.4–Principalcoordinatesanalysis(PCoA)representingthebetadiversityestimatedbytheunweightedUNIFRAC(4-A) andweightedUNIFRAC(4-B)method.Individualdatasetsarerepresentedasspheres,whicharecoloredaccordingtotheir samplesourceasfollows;Blue:metagenomicDNAsamplesextractedbythecommercialEpicentrekit,Red:metagenomic DNAsamplesextractedbythedevelopedchemicalprotocol.(4-A)Thethreeprincipalcoordinates(PC1–PC3)explain38.03, 24.49and16.84%,respectively.(4-B)Thethreeprincipalcoordinates(PC1–PC3)explain46.07,31.46and18.26%,respectively.

chemicalprotocolsyieldedhigherDNAthan theused

com-mercial kits.8 _The _three _protocols _used _in _this _study _(kit,

chemical, and gel puriﬁcation) achieved sufﬁcient removal

ofproteinsfrom the extractedmetagenomic DNAwiththe

absorbanceratiosat260/280nm>1.8.10,11_However,_the

high-estabsorbanceratios(260/280nm)wererecordedbythegel

puriﬁcationmethod, indicatingthatthe metagenomicDNA

extracted by this method had the lowest protein

concen-trations. The metagenomic DNA extracted by the kit and

the developed chemical protocol recorded the absorbance

ratiosat260/230nm<2,thisindicatedthepresenceofhumic

substances.9,11

Relatively high metagenomic DNA concentrations and

reasonableabsorbanceratiosdonotalwaysindicatethe

suit-abilityofextractedDNAforenzymaticreactions.Forinstance,

a previous study8 _investigated _ﬁve _different _metagenomic

DNA extraction methods but none of the extracted DNA

samples was suitable for PCR and/or restriction digestion

and thesamplesrequiredfurtherpuriﬁcationsteps.

(7)

ofthe metagenomic DNA, aspositive PCR results indicate

the absence of any impurities that may inhibit enzymatic

reactions.8,11_In_this_regard,_the_metagenomic_DNA_extracted

bythechemicalprotocoldevelopedinthisstudyshowed

pos-itivePCRresultsemphasizingtheefﬁciencyofthedeveloped

protocol.

The development of a sensitive, reproducible, and

pre-cisemetagenomicDNAextractionprotocolisessentialfora

successfulmetagenomicanalysis.Manyearlierstudieshave

useddifferentmoleculartechniquesasPCR,restriction

diges-tion, and cloning for testing the quality of the extracted

metagenomicDNA,8,9,11butnonereachedtheextentoftesting

the quality of the extracted DNA by applying the

down-streamanalysisoftheNGSoutputdata.Besides,metagenomic

DNA extraction protocols intended for the application in

NGSrequirehigh-qualitystandards.Speciﬁcally,NGSrequires

reproducible precise protocols that provide adequate read

length,numberofsequencespersample,andconsequently

thehighqualityofNGSdata.Inthisperspective,thecurrent

study adoptedaninitiative approachtoapplying NGSas a

qualitycontroltoolfordevelopingandoptimizingthe

metage-nomicDNAextractionprotocol.

Interestingly, the number of high-quality sequences

obtainedfrom themetagenomicDNAsamplesextractedby

thechemicalprotocolwas20%higherthanthatofthesamples

extracted by the Epicentre kit. This occurred as the

con-centrationof DNA extracted bythe chemical protocol was

signiﬁcantlyhigherthan thatextractedbytheEpicentrekit

(t-test,p-value=0.0425).Therewasnosigniﬁcantdifference

(Kruskal–Wallistest,p-value=0.682)intheobservedspecies

richness between the samplesextracted by bothmethods.

However,thesequencingdepth didnotcompletelycapture

the whole OTU richness in the kit samples, since,with a

highernumberofsequences inthechemicalprotocol

sam-ples,the curvewas stillrisingand didnotreachaplateau

inthe“observedspeciescurve”.Theimportanceofadequate

sequencingdepthforobtaininganaccurateestimationofthe

diversityinasamplewasstudiedpreviously,anditwas

indi-catedthatanincreasedsequencingdepthmarkedlyimproves

theestimatesofdiversity.19

Therewasnosigniﬁcantdifferenceinboththewithin

com-munitydiversity(alphadiversity)andthebetweencommunity

diversity(betadiversity)between thesamplesextracted by

bothmethods.Alltheseresultsindicateagreaterefﬁciency

of the developed chemical protocol to yield high-quality

metagenomicDNAcomparabletothatextractedbythe

well-establishedEpicentrekit.

When the tentative cost estimation of the optimized

method was attempted, it was found tobe approximately

0.125USDforprocessingonesample(SupplementaryTable

S1); this may beattributed to the use of low-cost

materi-als.Thepurifyingagentsusedinthedevelopedprotocolare

relatively inexpensive compared to other purifying agents

asanionresinsandhydroxyapatitecolumns.Incomparison

with any other commerciallyavailable kit, the cost of the

high-qualityDNAisolationfrom environmentalwater

sam-pleusingthedevelopedchemicalprotocolisremarkablylow

(processingonesampleusingEpicentrekitcostsabout6.85

USD).Ontheotherhand,thetimerequiredformetagenomic

DNAextractiondidnotvarymuchforeitherofthe studied

protocols; Epicentrekit and the developedchemical

proto-colrequired1.5hand2h,respectively,formetagenomicDNA

isolation.

In conclusion, acost and time-efﬁcientchemical

proto-col for the extractionof mDNA from environmentalwater

sampleshasbeendeveloped.Theefﬁcientrecoveryandhigh

qualityoftheextractedDNA usingthedevelopedchemical

protocolmakesitcompatiblewithhigh-resolutionmolecular

applicationsincludingnext-generationsequencing.This

pro-posesthedevelopedchemicalprotocoltobeagoldstandard

infuturemetagenomicstudiesinvestigatingalargenumber

ofsamples.

Authors’

contributions

MariamHassanandTamerEssamconceivedanddesignedthe

study.MariamHassanperformedthe experiments.Mariam

Hassan and Tamer Essamanalyzedthe data. Mariam

Has-san preparedtheﬁgures and illustrations. MariamHassan,

Tamer Essam,and SalwaMegahed draftedthe manuscript.

MariamHassanandTamerEssamwrotethepaperinﬁnal

for-mat.Allauthorsreadandapprovedtheﬁnalversionofthe

manuscript.

Funding

This work did notreceive any speciﬁc grant from funding

agenciesinthepublic,commercial,ornot-for-proﬁtsectors.

Conﬂicts

of

interest

Allauthorsdeclarethattheyhavenoconﬂictofinterest.

Acknowledgement

TheauthorsaredeeplygratefultoDr.AlexMira,Departmentof

GenomicsandHealth,CenterforAdvancedResearchinPublic

Health(CSISP),Valencia,Spain,forsequencingthesamples.

Appendix

A. Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,atdoi:10.1016/j.bjm.2018.03.002.

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