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We compared the variability between the replicates obtained at day one using OMNIgene.GUT and DNA/RNA Shield and found that the variability was similar both at the genus and at the phylum level. We also compared the overall variability pooling all 8 samples, and again the variability was comparable between the two buffers. Taxonomic quanJficaJon was slightly different between the two buffers for some taxonomical phyla, for example AcJnobacteria were increased the most significantly (P-val<0.0001) when using Omnigene.gut, and also Firmicutes whilst Cyanobacteria and Bacteroidetes were decreased compared to the samples preserved in DNA/RNA Shield. RESULTS MATERIAL & METHODS SELECTED KIT COMPONENTS Gut microbiome research is a very acJve area of research. It has over the last few years significantly increased our understanding of the beneficial or harmful role of the microbes residing in our guts. Luxia ScienJfic and IBBL have entered into a partnership to propose 1test1 TM , a test that compares your microbiome to the microbiome of a reference of French and Luxembourgish residents. The collecJon kit was designed to be suitable for home collecJon and transportaJon under uncontrolled temperature. To that end, we tested both the Omnigene TM kit (DNAGenotek) and the DNA/RNA Shield TM (Zymo Research/Ozyme), both stool collecJon kits contain a stabilizing soluJon which is of major importance to avoid preanalyJcal variaJons and also permit home collecJon. Previously IBBL opJmized and validated the combinaJon of the Omnigene TM kit with a magneJc beads-based DNA extracJon method (1). Based on the sequencing of the 16s V3-V4 region of a single donor and using IBBL’s automated pipeline, we found that the technical variability was suitable for commercial use and similar between both collecJon tubes. Given the smaller cost and simplified ease of use of the Zymo Research collecJon tube, we selected the DNA/RNA Shield TM collecJon tube as part of the home collecJon kit. u Outer waterproof envelope for shipping to clients u Customized pre labeled category B mailing box for shipment to IBBL u DNA/RNA Shield pre barcoded stool collecJon tube. Includes integrated spoon for easy sampling. u External tube envelope “shuble pouch TM ” includes 15 ml absorbant material to prevent leakage. u Stool collecJon paper suitable for IVD use and CE marked. Larger size ensures best results. u Customised instrucJon sheet for clearer instrucJons. CONCLUSION RAW DATA INTRODUCTION ABSTRACT Laboratory work REFERENCES & ACKNOWLEDGMENT Pre- analyJcs such as sample collecJon methods are essenJal to ensure the accuracy and reproducibility of human microbiome studies. The gut microbiome can be analysed by sequencing of the 16S rRNA gene or by shotgun sequencing, of a stool sample. Historically to stabilize the stool’s bacterial content, freezing was used. However, stool freezing is not a suitable opJon for home collecJon studies. As a result, the use of stabilizaJon soluJons to stabilize the DNA and RNA are needed. Voigt et al. 2015 performed metagenomic analysis on faecal samples and invesJgated fresh frozen collecJon compared to RNALater. They concluded that RNALater was a suitable alternaJve to fresh freezing. Choo et al. 2015 also compared fresh freezing to RNAlater, OMNIgene.GUT (DNAGenotek) and Tris-EDTA. They found that, OMNIgene.GUT resulted in the least alteraJon compared to the frozen sample. Hill et al. 2016 compared fresh extracJon to OMNIgene.GUT, they reported a greater variability in low diversity samples. As a result of several independent publicaJons supporJng it’s suitability, OMNIgene.GUT is likely the most widely used ambient stool collecJon method. More recently, Zymo research launched a stool collecJon tube with the DNA/RNA shield as a stabilizaJon buffer. As no independent evaluaJon was available, we decided to compare the stability of a stool sample collected using OMNIgene. GUT versus Zymo’s DNA/RNA shield. 1: Luxia ScienJfic, 3 Rue de Tournezy, 77590 Bois Le Roi, France 2: IBBL, 1, rue Louis Rech, L-3555 Dudelange, Luxembourg 1. Mathay et al, 2015. Biopreserv Biobank. 2015 Apr;13(2):79-93. Method opJmizaJon for fecal sample collecJon and fecal DNA extracJon. 2. Voigt et al. 2015. Genome Biology. 16(1). Temporal and technical variability of human gut metagenomes. 3. Hill et al. 2016. Microbiome. 4(1). Effect of room temperature transport vials on DNA quality and phylogeneJc composiJon of faecal microbiota of elderly adults and infants. 4. Choo et al. 2015. ScienJfic Reports. 5. Sample storage condiJons significantly influence faecal microbiome profiles. The authors would like to thank Fay Betsou for her input and support. DESIGN OF A STOOL COLLECTION KIT FOR HOME BASED MICROBIOME RESEARCH 16 samples from single donor over 3 days We found that DNA/RNA Shield by Zymo research is simpler of use, thanks to the integrated spoon instead of a spatula and has comparable performance in terms of stabilisaJon over two days. The overall variability which also includes the technical variability of DNA extracJon and sequencing is low and makes it suitable for commercialisaJon. The assembly of a dedicated stool collecJon kit has resulted in a category B kit with the following benefits: • Highest quality standards • Simplicity of use • Cost efficiency Our collecJon kit is planned for CE marking end of 2018. Alessandra Cervino *1 , Ivo Ugrina 1 , Ignacio Sanchez 2 , Conny Mathay 2 , Angela Hogan 2 , Gaël Hamot 2 , Eleoheria Charalambous 2 and Wim Ammerlaan 2 *acervino@luxia-scien3fic.com DNA RNA Shield Stool Day 3 Stool Day 2 DNA RNA Shield DNA RNA Shield Omnigene.gut Omnigene.gut Omnigene.gut DNA RNA Shield DNA RNA Shield DNA RNA Shield Omnigene Omnigene Omnigene Stool Day 1 Shipped Day 1 Shipped Day 2 Omnigene Omnigene DNA RNA Shield DNA RNA Shield DNA/RNA Shield OMNIgene.GUT BioinformaJcs analysis Fastq files were imported and analysed by Luxia ScienJfic using Qiime.2. Taxonomical analysis was performed at the phylum and at the genus level. StaJsJcal analysis and graphs were generated using Rstudio. 1 – Stool DNA extracBon In house validated method using Chemagic DNA Blood Kit Special 4ml (Perkin-Elmer ref. CMG-1074) combined with Chemagic Stool Lysis Buffer (Perkin-Elmer, ref. SEB). ExtracJon with MSM I 24-rod head and further processed by Janus liquid handler (Perkin Elmer) (ref.1) 2 - DNA QuanBficaBon ISO 17025 accredited method based on Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher) read on a Synergy MX (Biotek) 3 - 16S Sequencing ISO 17025 accredited method based on Illumina 16S Metagenomic Sequencing Library PreparaJon and subsequent sequencing on the MiSeq (Illumina) Plus d'infos sur DNA/RNA Shield, nous contacter

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Page 1: DESIGN OF A STOOL COLLECTION KIT FOR HOME BASED …

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*[email protected]

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)

Plus d'infos sur DNA/RNA Shield, nous contacter

www.ozyme.fr/contact
www.ozyme.fr/contact
www.ozyme.fr/contact

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