WecomparedthevariabilitybetweenthereplicatesobtainedatdayoneusingOMNIgene.GUTandDNA/RNAShieldandfoundthatthevariabilitywassimilarbothatthegenusandatthephylumlevel.Wealsocomparedtheoverallvariabilitypoolingall8samples,andagainthevariabilitywascomparablebetweenthetwobuffers.TaxonomicquanJficaJonwasslightlydifferentbetweenthetwobuffersforsometaxonomicalphyla,forexampleAcJnobacteriawereincreasedthemostsignificantly(P-val<0.0001)whenusingOmnigene.gut,andalsoFirmicuteswhilstCyanobacteriaandBacteroidetesweredecreasedcomparedtothesamplespreservedinDNA/RNAShield.

RESULTS

MATERIAL&METHODS

SELECTEDKITCOMPONENTS

GutmicrobiomeresearchisaveryacJveareaofresearch.Ithasoverthelastfewyearssignificantlyincreasedourunderstandingofthebeneficialorharmfulroleofthemicrobesresidinginourguts.LuxiaScienJficandIBBLhaveentered intoapartnership topropose1test1TM,a test thatcomparesyourmicrobiometo themicrobiomeofareference of French and Luxembourgish residents. The collecJon kit was designed to be suitable for homecollecJonandtransportaJonunderuncontrolledtemperature.Tothatend,wetestedboththeOmnigeneTMkit(DNAGenotek)andtheDNA/RNAShieldTM(ZymoResearch/Ozyme),bothstoolcollecJonkitscontainastabilizingsoluJon which is of major importance to avoid preanalyJcal variaJons and also permit home collecJon.PreviouslyIBBLopJmizedandvalidatedthecombinaJonoftheOmnigeneTMkitwithamagneJcbeads-basedDNAextracJon method (1). Based on the sequencing of the 16s V3-V4 region of a single donor and using IBBL’sautomatedpipeline,wefoundthatthetechnicalvariabilitywassuitableforcommercialuseandsimilarbetweenbothcollecJontubes.GiventhesmallercostandsimplifiedeaseofuseoftheZymoResearchcollecJontube,weselectedtheDNA/RNAShieldTMcollecJontubeaspartofthehomecollecJonkit.

u Outerwaterproofenvelopeforshippingtoclients

u CustomizedprelabeledcategoryBmailingboxforshipmenttoIBBL

u DNA/RNAShieldprebarcodedstoolcollecJontube.Includesintegratedspoonforeasysampling.

u Externaltubeenvelope“shublepouchTM”includes15mlabsorbantmaterialtopreventleakage.

u StoolcollecJonpapersuitableforIVDuseandCEmarked.Largersizeensuresbestresults.

u CustomisedinstrucJonsheetforclearerinstrucJons.

CONCLUSION

RAWDATAINTRODUCTIONABSTRACT

Laboratorywork

REFERENCES&ACKNOWLEDGMENT

Pre- analyJcs such as sample collecJon methods are essenJal to ensure the accuracy and reproducibility of humanmicrobiomestudies.Thegutmicrobiomecanbeanalysedbysequencingofthe16SrRNAgeneorbyshotgunsequencing,ofastoolsample.Historicallytostabilizethestool’sbacterialcontent,freezingwasused.However,stoolfreezingisnotasuitableopJonforhomecollecJonstudies.Asaresult,theuseofstabilizaJonsoluJonstostabilizetheDNAandRNAareneeded.Voigtetal.2015performedmetagenomicanalysisonfaecalsamplesandinvesJgatedfreshfrozencollecJoncomparedtoRNALater.TheyconcludedthatRNALaterwasasuitablealternaJvetofreshfreezing.Chooetal.2015alsocomparedfreshfreezingtoRNAlater,OMNIgene.GUT(DNAGenotek)andTris-EDTA.Theyfoundthat,OMNIgene.GUTresultedintheleastalteraJoncomparedtothefrozensample.Hilletal.2016comparedfreshextracJontoOMNIgene.GUT,theyreportedagreater variability in low diversity samples. As a result of several independent publicaJons supporJng it’s suitability,OMNIgene.GUTislikelythemostwidelyusedambientstoolcollecJonmethod.Morerecently,ZymoresearchlaunchedastoolcollecJontubewiththeDNA/RNAshield asastabilizaJonbuffer.AsnoindependentevaluaJonwasavailable,wedecidedtocomparethestabilityofastoolsamplecollectedusingOMNIgene.GUTversusZymo’sDNA/RNAshield.

1:LuxiaScienJfic,3RuedeTournezy,77590BoisLeRoi,France2:IBBL,1,rueLouisRech,L-3555Dudelange,Luxembourg

1. Mathayetal,2015.BiopreservBiobank.2015Apr;13(2):79-93.MethodopJmizaJonforfecalsamplecollecJonandfecalDNAextracJon.

2. Voigtetal.2015.GenomeBiology.16(1).Temporalandtechnicalvariabilityofhumangutmetagenomes.3. Hilletal.2016.Microbiome.4(1).EffectofroomtemperaturetransportvialsonDNAqualityandphylogeneJc

composiJonoffaecalmicrobiotaofelderlyadultsandinfants.4. Chooetal.2015.ScienJficReports.5.SamplestoragecondiJonssignificantlyinfluencefaecalmicrobiomeprofiles.

TheauthorswouldliketothankFayBetsouforherinputandsupport.

DESIGNOFASTOOLCOLLECTIONKITFORHOMEBASEDMICROBIOMERESEARCH

16samplesfromsingledonorover3days

WefoundthatDNA/RNAShieldbyZymoresearchissimplerofuse,thankstotheintegratedspooninsteadofaspatulaandhascomparable performance in terms of stabilisaJon over two days. The overall variability which also includes the technicalvariabilityofDNAextracJonandsequencingislowandmakesitsuitableforcommercialisaJon.TheassemblyofadedicatedstoolcollecJonkithasresultedinacategoryBkitwiththefollowingbenefits:• Highestqualitystandards• Simplicityofuse• CostefficiencyOurcollecJonkitisplannedforCEmarkingendof2018.

AlessandraCervino*1,IvoUgrina1,IgnacioSanchez2,ConnyMathay2,AngelaHogan2,GaëlHamot2,EleoheriaCharalambous2andWimAmmerlaan2*acervino@luxia-scien3fic.com

DNARNAShield

StoolDay3StoolDay2

DNARNAShield

DNARNAShield

Omnigene.gut

Omnigene.gut

Omnigene.gut

DNARNAShield

DNARNAShield

DNARNAShield

Omnigene

Omnigene

Omnigene

StoolDay1

ShippedDay1ShippedDay2

Omnigene Omnigene

DNARNAShield DNARNAShield

DNA/RNAShield OMNIgene.GUT

BioinformaJcsanalysis

Fastq files were imported andanalysed by Luxia ScienJfic usingQiime.2. Taxonomical analysiswasperformedatthephylumandat the genus level. StaJsJcalana lys i s and graphs weregeneratedusingRstudio.1–StoolDNAextracBon

InhousevalidatedmethodusingChemagicDNABloodKitSpecial4ml(Perkin-Elmerref.CMG-1074)combinedwithChemagicStoolLysisBuffer(Perkin-Elmer,ref.SEB).ExtracJonwithMSMI24-rodheadandfurtherprocessedbyJanusliquidhandler(PerkinElmer)(ref.1)

2-DNAQuanBficaBonISO17025accreditedmethodbasedonQuant-iT™PicoGreen™dsDNAAssayKit(ThermoFisher)readonaSynergyMX(Biotek)

3-16SSequencingISO17025accreditedmethodbasedonIllumina16SMetagenomicSequencingLibraryPreparaJonandsubsequentsequencingontheMiSeq(Illumina)

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