Automated Nucleic Acid Purification from Diverse Sample types using dedicated Microbiome kits on the QIAcube

Sample to Insight

Automated nucleic acid purification from diverse sample types using dedicated microbiome kits on the QIAcube.

Patrick Smith, PhD

Scientist R&D QIAGEN GmbH

[email protected]

Sample to Insight

Overview

Introduction

◦ Microbiome

◦ Inhibitor Removal Technology® (IRT)

QIAcube

◦ Introduction to nucleic acid purification with the QIAcube

Semi-automated nucleic acid isolation

◦ DNA isolation using dedicated microbiome kits

◦ RNA isolation using dedicated microbiome kits

Sample to Insight workflow

◦ DNA isolation with QIAamp PowerFecal kit on the QIAcube

◦ NGS library preparation (QIASeq)

◦ Bioinformatics with CLC Microbial Genomics Workbench

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Overview

Introduction

◦ Microbiome


QIAcube









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Microbiome: Definition and background

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What does “microbiome” mean?

“The microbiome is defined as the collective genomes of the

microbes (composed of bacteria, bacteriophage, fungi, protozoa,

and viruses) that live inside and on the human body.”

-NIH, 2012

Microbiota refers to the collection of microbial organisms that

inhabits a certain environment

Metagenomics is the study of the collective genomes of

microorganisms from a sample without cultivation (Lederberg and

McCray 2001, The NIH HMP Working Group)

Kuczynski et al. Nature Reviews Genetics 13, 47-58 (January 2012)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792171/

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Human Microbiome Project

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Microorganisms cluster by body siteCataloguing efforts by the NIH Human Microbiome Project suggest:

• ~10,000 organisms live with us

• Second genome: outnumber

our own genes 150:1

Identifying microbiota in healthy individuals revealed:

• Different body sites have

unique communities

• Race, age, gender, weight or

ethnicity have an effect

1 Hoffmann A.R., et al. “The Microbiome: The Trillions of Microorganisms That Maintain Health and Cause Disease in Humans and Companion Animals.” Vet Pathol. 20152 http://commonfund.nih.gov/hmp/index 3 Structure, function and diversity of the health human microbiome. The Human Microbiome Project Consortium. Nature, 486, 207-214 (14 June 2012). doi: 10.1038/nature11234

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Earth Microbiome Project

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Multidisciplinary effort to survey the microbial composition of diverse environments across the globe:

• Aims to process 200,000 samples from these different biomes

• Generate a database of microbes and their gene products

• Will greatly enhance our understanding of the roles microbes play in ecology

• Will expand our understanding of microbial metabolism and gene products

• Estimates of bacterial diversity1:

• 160 distinct types of bacteria in 1 ml of ocean water

• 6,400 – 38,000 types of bacteria in 1 gram of soil

These are just estimates for bacteria alone; one still needs to consider viruses, archaea

and fungi

1Curtis, T. P.; Sloan, W. T.; Scannell, J. W. (2002). "Estimating prokaryotic diversity and its limits". Proceedings of the

National Academy of Sciences 99 (16): 10494–10499.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC124953

Sample to Insight

About MO BIO Laboratories:

• 22 year-old Carlsbad, CA molecular biology company

– Privately owned until acquisition by QIAGEN in November 2015

– World leader in DNA and RNA isolation tools for microbiome analysis

• Products now fully integrated with QIAGEN

– 9 kits available on QIAcube

– Custom protocols available

• Products sold in 90+ countries

MO BIO’s sample prep solutions

Sample to Insight

Projects using MO BIO Kits with Inhibitor Removal Technology®

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Molecular technologies for microbial community analysis

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16S rRNA gene

sequencingTotal DNA sequencing

(shotgun)

Bacteria and

Archaea

Fungus

/ Yeast

Viruses Gene content

RNA expression profiling

(transcriptomics)

Gene expressionMetabolite, protein

characterization

Mass spectroscopy

(metabolomics &

proteomics)

Identify relative frequencies and pathways

microbiome sample

Extract DNA Extract RNA Extract proteins & small

molecules

What organisms are present and

what is their relative abundance? What are the functions of the community?

Sample to Insight

Sample preparation requirements for successful microbiome studies

There are several areas where sample prep inefficiencies can bias a

microbiome/metagenomics study:

• Co-purification of small molecule inhibitors of amplification reactions (e.g., PCR)

Decreases efficiency of amplification or can inhibit library prep reactions

• Insufficient cell lysis

Bias downstream analysis toward ‘the easily disrupted’ population(s)

• Reproducible isolation of high quality nucleic acids

• Poor nucleic acid quality, extensive shearing

• Insufficient solubilization of analyte(s) of interest/separation from interacting cellular

components

• Unintended precipitation of nucleic acids via complexation with matrix-derived

metals, bioactive amines

• Insufficient homogenization of sample matrix (to dislodge/disrupt cell:substrate

interactions)

Sample to Insight

High Inhibitors

Difficult Lysis

MO BIO’s sample prep solutions

Easy DifficultLysis

Inh

ibit

ors

Low

High

Blood, animal

tissue & cells

Pure microbial

cultures

Soil microbesLeaf

tissue

Stool & gut

microbes

Biofilm

FFPE

Tissue

Food cultures

Sample to Insight

Sample-derived PCR/RT-PCR inhibitors

In the process of breaking open cells to

release nucleic acids, amplification

inhibitors are also released.

*Other examples for additional matrices given in Rådström, P. et al. (2004) Pre-PCR processing: Strategies to

generate PCR-compatible samples. Mol. Biotechnol. 26, 133–46.

Inhibitors include humic/fulvic acids in soil,

polysaccharides/polyphenolics in plants

and bile, bilirubin and heme in stool*

Sample to Insight

No IRT

IRT Method

IRT MethodNo IRT

No IRT

IRT

No IRT

No IRT

IRT

IRT

Why contamination removal is important

Sample to Insight

Low levels of contaminating inhibitors can lead to false negatives / aberrant amplification

IRT + + - -

+ IRT

- IRT

Samples 260/280 260/230

IRT 1.91 2.03

IRT 1.92 1.99

No IRT 1.87 1.84

No IRT 1.85 1.53

Low levels of inhibitors can also inhibit other enzymatic reactions commonly found in WGS library prep kits

Sample to Insight

Overview

Introduction

◦ Microbiome


QIAcube









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QIAcube: An in-depth look

QIAcube in-depth

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Sample to Insight

QIAcube – Technical overview

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Sample to Insight

QIAcube: Purification

QIAcube in NGS, Hilden, November 2015 18

Sample NGS run InsightSample prep

on QIAcube

▪ Low throughput (12 samples per run) automated nucleic acid purification

▪ DNA, RNA and protein purification

▪ No change from manual spin-column procedure

▪ Benchtop, plug and play

▪ Elimination of manual processing steps

▪ Standardized protocols ensure control

▪ 130 standardised protocols

▪ Customisable protocols also available

Sample to Insight

QIAcube: Purification setup

QIAcube in NGS, Hilden, November 2015 19

Load the sample Load the buffers Load the adaptors

Checking number and types of tips Checking after tip uptake

Verifying loading of centrifuge Checking for sufficient buffer volumesChecking samples in loaded shaker

Sample to Insight

QIAcube - Applications

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Genomic DNA from human samples

QIAamp DNA Blood Mini Kit

QIAamp DNA Mini Kit

QIAamp DNA Stool Mini Kit

QIAamp DNA Micro Kit

QIAamp MinElute Media Kit

QIAamp DSP DNA Blood Mini kit [IVD]

QIAamp DSP DNA Mini kit [IVD]

QIAamp Circulating NA

PAXgene Tissue DNA Kit

QIAamp Fast DNA Stool Mini Kit

Forensic samples

QIAamp DNA Investigator Kit

Differential Wash protocol (custom.)

Viral DNA and RNA purification

QIAamp MinElute Virus Spin Kit

QIAamp Viral RNA Mini Kit

QIAamp DSP MinElute Virus Spin Kit

[IVD]

QIAamp DSP Virus Mini kit [IVD]

QIAamp DSP Viral RNA Mini kit [IVD]

QIAamp cador Pathogen Mini

FFPE samples

RNeasy Plus Universal Kit

RNeasy FFPE Kit

QIAamp DNA FFPE Kit

AllPrep DNA/RNA FFPE

miRNeasy FFPE

Plant or food samples

DNeasy Plant Mini Kit

RNeasy Plant Mini Kit

DNeasy mericon Food Kit

Bisulfite conversion and cleanup

EpiTect Bisulfite Kit

EpiTect Plus DNA Bisulfite Kit

EpiTect Fast DNA Bisulfite

Protein purification and serum depletion

Ni-NTA Spin Kit

Qproteome Albumin/IgG Depletion Kit

Qproteome Murine Albumin Depletion

Kit

Qproteome Total Glycoprotein Kit

Qproteome O-Glycan Glycoprotein Kit

Genomic DNA from animal samples

DNeasy Blood & Tissue Kit

Plasmid DNA purification

QIAprep Spin Miniprep Kits

DNA cleanup

QIAquick PCR Purification Kit

QIAquick Gel Extraction Kit

QIAquick Nucleotide Removal Kit

MinElute PCR Purification Kit

MinElute Reaction Cleanup Kit

MinElute Gel Extraction Kit

NGS specific Kits

GeneRead rRNA Depletion Kit

GeneRead Size Selection

GeneRead DNA Library I Core Kit

GeneRead DNA Library L Core Kit

Multiple analyte purification

AllPrep DNA/RNA Kits

AllPrep DNA/RNA FFPE Kit

AllPrep DNA/RNA Micro Kit

AllPrep DNA RNA miRNA Universal Kit

RNA purification from a range of

samples

RNeasy Mini Kit

RNeasy MinElute Cleanup Kit

RNeasy Plus Mini Kit

RNeasy Micro Kit

RNeasy Plus Micro Kit

RNeasy Lipid Tissue Mini Kit

RNeasy Fibrous Tissue Kit

RNeasy Protect Bacteria Mini Kit

RNeasy Protect Animal Blood Kit

miRNeasy Protect Animal Blood Kit

miRNeasy Serum/Plasma Kit

RNeasy Protect Animal Blood Kit

PAXgene Blood RNA Kit [IVD]

PAXgene Blood miRNA Kit

QIAamp RNA Blood Mini Kit

RNeasy Plus Universal Kit

RNeasy Microarray Tissue

Microbiome

MOBIO kits now available

on the QIAcube!

Sample to Insight

Overview

Introduction

◦ Microbiome


QIAcube









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• All in one protocol for sample preparation and removal of inhibitors

• Sample material:

• Inherently rich in inhibitors: Mammalian stool (human, mouse, cow, horse), bird

stool, environmental samples (water, soil, biofilm, plant)

• Downstream application:

• Whole metagenome shotgun sequencing

• 16S rRNA gene sequencing

• qPCR, PCR genotyping

Microbial

Cell Lysis

Inhibitor

Removal

Microbial

DNA

Isolation

AnalysisSample

Collection

Nucleic Acid Isolation with Dedicated Microbiome Kits

Mechanical lysis Patented Inhibitor

Removal Technology

QIAamp PowerFecal Kit

Sample to Insight

Nucleic Acid Isolation with Dedicated Microbiome Kits

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• Following mechanical lysis the supernatant is loaded directly into the QIAcube

• QIAcube performs all inhibitor removal, DNA binding, wash and elution steps

• Hands free high quality DNA is isolated in ~45-60 min, which can be used

immediately in downstream applications

Microbial

Cell Lysis

Inhibitor

Removal

Microbial

DNA

Isolation

AnalysisSample

Collection

Performed Manually Performed by QIAcube

Sample to Insight

Environmental Research

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Soil

Distinct, diverse microbial species populate the soil of temperate, desert, tropical and arctic

regions

Soil biodiversity is influenced pH and the chemical composition of soil organic matter within the

soil environment

Isolation of nucleic acids from soil is complicated by the large amounts of humic acids

contained within

DNeasy PowerSoil Kit

Sample to Insight


High quality DNA was isolated from a variety of samples including landfill, compost

and horse manure using the DNeasy PowerSoil Kit compared with two other

suppliers. All isolation were performed following the manufacturer’s protocols.

High yield, intact genomic DNA

Sample to Insight


Complete removal of inhibitors allows for more accurate PCR and qPCR analyses

and reduces false negatives. PCR was performed using total bacterial primers on

1 ul of undiluted DNA.

More accurate PCR analysis

Sample to Insight

DNeasy PowerSoil Kit – DNA Yield

manual extraction

QIAcube extraction

A B

C D

Estuary Compost

manual extraction

QIAcube extraction

manual extraction

QIAcube extraction

A B

C D

Estuary Compost

manual extraction

QIAcube extraction

• 250mg of either Estuary or compost soil

was used as input material

• There were no differences in yield or

quality between manual and QIAcube

protocol

• Purified DNA had high 260/280 and

260/230 ratios

• Average 260/280 = 1.8

• Average 260/230 = 1.7

Sample to Insight

DNeasy PowerSoil Kit – PCR Inhibition

• PCR inhibition was measured using

QIAGEN QuantiFast Pathogen PCR + IC

Kit

• 5-10ul of DNA eluate was added to each

rxn

• If inhibitors are present change in Ct value

is typically between 3 and 15

• DNA is ready for immediate use in

downstream applications

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

Ct

valu

e

Estuary

manual extraction QIAcube estraction

05

10152025303540

Ct

va

lue

Compost

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Water

Distinct, diverse microbial species populate the water of Oceans, Lakes, Rivers, Marshes and

arctic regions

Water contains large amounts of humic acids, heavy metals, chemical and biological waste

QIAcube automation of the DNeasy PowerWater Kit

Carlsbad Lagoon, Carlsbad, Ca

Pacific Ocean, Encinitas, Ca

Sample to Insight

DNeasy PowerWater Kit – DNA Yield

• 75ml of lagoon water or 150ml Ocean

water as input material



protocol


260/230 ratios

• Average 260/280 = 1.8

• Average 260/230 = 1.5

0

0,5

1

1,5

2

2,5

3

3,5D

NA

yie

ld (

ug)

Lagoon water

manualprotocol

QIAcubeprotocol

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

0,18

Ocean water

DN

A y

ield

(u

g)

Sample to Insight

DNeasy PowerWater Kit – PCR Inhibition

• PCR inhibition was measured using

QIAGEN QuantiFast Pathogen PCR + IC

Kit

• 5-10ul of DNA eluate was added to each

rxn

• If inhibitors are present change in Ct value

is typically between 3 and 15

• DNA is ready for immediate use in

downstream applications

Manual extraction QIAcube extraction

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 control

Ct

va

lue

Lagoon Water

Manual

QIAcube

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Biofilm

Biofilms are diverse mixtures of microbes that can be very difficult to lyse

Microbial Mats, Stream/Lagoon Rocks, Gypsum Crust, Sink Pipe

Lysis difficulty is due to the formation of extracellular polymeric substances

DNA, Proteins and polysaccharides

QIAcube automation of the DNeasy PowerBiofilm Kit

Sample to Insight

DNeasy PowerBiofilm Kit – DNA Yield

• 0.25mg of concrete Biofilm



protocol


260/230 ratios

• Average 260/280 = 1.9

• Average 260/230 = 1.9

0

1

2

3

4

5

6

Concrete Biofilm

DN

A Y

ield

(u

g)

ManualProtocol

QIAcubeProtocol

Manual Extraction QIAcube Extraction

Sample to Insight

DNeasy PowerBiofilm Kit – PCR Inhibition

• No Inhibition in either the manual or QIAcube protocols


0

5

10

15

20

25

30

35

40

1 2 3 4 5 Control

Ct

Val

ue

Concrete Biofilm

Manual Protocol

QIAcube Protocol

Sample to Insight

Biological Research

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RNA from human and animal stool

Can be difficult to lyse without introducing microbial biases

Can contain high amount of inhibitors, heme, bile acids, polysaccharides

Intact high quality RNA can be difficult to obtain due to the amount of RNases and other

degrading enzymes present in stool

QIAcube automation of the RNeasy PowerMicrobiome Kit

Sample to Insight

RNeasy PowerMicrobiome Kit – DNA Yield

• 200mg of human stool as input

• Isolated a high yield of high quality RNA

• There were no differences in yield or quality between manual and QIAcube

protocol

0

5

10

15

20

25

30

35

40

45

50

Human Stool

RN

A Y

ield

(u

g)

0

0,5

1

1,5

2

2,5

3

Human Stool

26

0/2

30

nm

Ra

tio

0

0,5

1

1,5

2

2,5

Human Stool

26

0/2

80

nm

Ra

tio

Manual Protocol QIAcube Protocol

Sample to Insight

RNeasy PowerMicrobiome Kit– PCR Inhibition

• Comparable Inhibition in either the manual or QIAcube protocols


0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 No_IRT Control

Ct

Val

ue

Manual Protocol

QIAcubeProtocol

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Overview

Introduction

◦ Microbiome


QIAcube









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Semi-automated DNA Isolation With QIAamp PowerFecal Kit

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Performed Manually Performed by QIAcube

Microbial

Cell Lysis

Inhibitor

Removal

Microbial

DNA

Isolation

Library

Prep

Sample

Collection

• Human Stool, N = 10

• Aged samples:

female (77), female

(78), female (74),

male (77), male (65)

• Young samples:

female (28), female

(49), male (28), male

(28), male (37)

• Additional Metadata

– race, diet

• Use samples directly

for 16s and whole

genome sequencing

applications

• Analyze data with

CLC Microbial

Genomics Pro Suite

Sample to Insight

Semi-automated DNA Isolation With QIAamp PowerFecal Kit

40

0

5

10

15

Old Young

DN

A (

ug

)

Isolation of high quality DNA

• 200mg human stool lysed in PowerBead tubes for

10 minutes using a vortex adapter

• Supernatant was then placed in QIAcube for DNA

isolation

• Agarose gel electrophoresis shows isolation of

high molecular weight genomic DNA with minimal

degradation

• For each sample, several μg of DNA was isolated

as measured by a flourescence based assay

Sample to Insight

16S rRNA gene library prep

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Methods for microbiome analyses:

16s rRNA gene sequencing for community composition

Workflow:

Mixed sample DNA isolation —

host and microbes16S rDNA amplification

Ashelford et al., Appl. Environ. Microbiol. 71 7724-7736 (December 2005)

Community composition

Diversity within and

between samples

Amplicon

SequencingData

Analysis

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• 16S PCR amplicons – 22 cycles

Young SamplesOld Samples Blanks

-500

-300

-1000

bp

Inhibitor removal from DNA allows efficient amplification/yield of V4 16S region

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• 50 ng inhibitor free DNA in 16s rDNA PCR

• Modified 515f PCR primer and 806r PCR primer

• Addition of adapters with indices using QIAseq 1-step Amplicon kit

• 2x250 bp on Illumina MiSeq

• 500 ng amplicon as input, all libraries > 10 nM final concentration

Sample to Insight

Whole Genome Sequencing library prep

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Purified

gDNA(1ng

-1μg)

Single

Tube FX

Rxn

(60min)

Purification

sequencin

g

Adapter

ligation

(45 min)

• Accepts wide

range of input

DNA

• Easily

customizable

fragment sizes

• Adapter ligation in

a single step, with

dual indices

• Fully compatible

with Illumina

sequencing

Sample to Insight

16S and WGS Sequencing Statistics

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• 16s - 2 x 250 bp with Illumina MiSeq

◦ Generated > 22 million paired end reads

◦ ~ 90% reads passed quality filter, %>Q30 85

◦ ~ 88% of reads were assigned to an index

◦ Of reads identified, 9 of 10 samples were between 9-11% of total reads

• WGS – 2 x 250bp with illumina MiSeq

◦ Generated > 27 million paired end reads

◦ 97% reads passed quality filter, %>Q30 88

◦ 88% of reads were assigned to a dual index

◦ Of reads identified, 9 of 10 samples were between 8-10% of total reads

• DNA isolation through library prep and sequencing worked great!

Sample to Insight

QIAGEN Microbial Genomics Pro Suite

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From Data to Discovery

• Integrated analytics deliver research continuity

◦ All analytics for microbial genomics and metagenomics comes fully integrated

◦ Fully Scalable with data and sample-metadata management included

• Focus on what matters

◦ Streamlined workflow allows users to focus on interpretation of results

• High performance algorithms

◦ Designed to save time and compute resources

◦ Accessible to bioinformatics experts and non-bioinformaticians alike

Sample to Insight


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Powered by Quality Components

• Microbial genmoics Pro Suite Expands upon CLC

genomic Workbench

• Plugins and modules add a layer of specialization

◦ Download CLC microbial genomics module,

CLC genom finishing module,

MetaGeneMarkPlugin

• Upload data and with customizable workflows data

is just a few clicks away

• Easy to use Graphical User Interface

• Quick and helpful customer service

Sample to Insight


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16S and WGS workflows

Sample to Insight


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16S OTU and taxonomic assignment workflow

• Includes quality filtering

• For OTU picking, sequences were mapped

against the greengenes data base and clustered

at 97% identity

• Unmapped sequences were then clustered de

novo

• Results shown are summarized at the genus

level

• Highly customizable outputs

• Differential Abundance analysis (not shown)

Sample to Insight


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Alpha and Beta Diversity Workflow

• Young individuals have a higher alpha diversity

compared with old as measured by observed

OTUs

• Other metrics include, Shannon and Simpson

Diversity, phylogenetic diversity

• Highly customizable output

• Beta diversity: unweighted UniFrac Analysis

• Young and old gut communities are significantly

different. P = 0.047 Permanova analysis,

corrected for multiple comparisons

• Other metrics include weighted UniFrac,

gneralized UniFrac, Bray-Curtis, Euclidean

• Highly customizable outputs

Sample to Insight


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WGS read mapping and Taxonomic assignment workflow

• Includes quality filtering and read merging

• The taxonomic profiler maps each read

against the chosen database

• Output is an abundance table including

graphical representation


• Graph shown at left is summarized at the

genus level

• CLC Microbial Genomics Pro Suite can also

alpha and beta diversity analyses (not shown)

Sample to Insight


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WGS De novo assembly and Functional assignment workflow

• Includes quality filtering

• De novo assembly of reads, identification and

annotation of genes and coding sequences

• Perform functional and statistical analyses


• Samples are clustered by functional similary

using Euclidean distances

• CLC Microbial Genomics Pro Suite can also

perform taxonomic assignment (not shown)

Sample to Insight


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WGS De novo assembly and Functional assignment workflow

Name

Max group

mean

Log₂ fold

change Fold change P-value FDR p-value

0008452 // RNA ligase activity 151.2 -1.64 -3.12 0.000216912 0.27

0071804 // cellular potassium ion transport 47 5.04 32.84 0.000765205 0.27

0071805 // potassium ion transmembrane transport 47 5.04 32.84 0.000799437 0.27

0016407 // acetyltransferase activity 1229.8 -0.38 -1.30 0.001037548 0.27

0016410 // N-acyltransferase activity 1039 -0.43 -1.35 0.001044564 0.27

0046353 // aminoglycoside 3-N-acetyltransferase activity 22.6 -7.56 -188.37 0.001054322 0.27

0034069 // aminoglycoside N-acetyltransferase activity 22.6 -7.56 -188.37 0.001060815 0.27

0008080 // N-acetyltransferase activity 1039 -0.43 -1.35 0.001068129 0.27

0008234 // cysteine-type peptidase activity 615.2 0.57 1.48 0.00138698 0.28

0006276 // plasmid maintenance 145.2 3.68 12.85 0.001412554 0.28

0010393 // galacturonan metabolic process 16.8 -7.10 -137.19 0.002238434 0.33

0016837 // carbon-oxygen lyase activity, acting on polysaccharides 16.8 -7.10 -137.19 0.002247484 0.33

0045490 // pectin catabolic process 16.8 -7.10 -137.19 0.002280259 0.33

0045488 // pectin metabolic process 16.8 -7.10 -137.19 0.002313312 0.33

0047487 // oligogalacturonide lyase activity 15.8 -7.02 -129.63 0.002642413 0.35

0006787 // porphyrin-containing compound catabolic process 15.2 -6.93 -121.66 0.003588363 0.38

0046149 // pigment catabolic process 15.2 -6.93 -121.66 0.003699807 0.38

0033015 // tetrapyrrole catabolic process 15.2 -6.93 -121.66 0.00381645 0.38

0015994 // chlorophyll metabolic process 15.2 -6.93 -121.66 0.003925426 0.38

0047746 // chlorophyllase activity 15.2 -6.93 -121.66 0.003935538 0.38

0015996 // chlorophyll catabolic process 15.2 -6.93 -121.66 0.003991415 0.38

0033926 // glycopeptide alpha-N-acetylgalactosaminidase activity 10.2 -7.30 -157.15 0.00434989 0.39

0036211 // protein modification process 1168.6 -0.43 -1.34 0.004493368 0.39

0006464 // cellular protein modification process 1168.6 -0.43 -1.34 0.004604395 0.39

0019439 // aromatic compound catabolic process 891.6 -0.51 -1.42 0.006839066 0.53

1901361 // organic cyclic compound catabolic process 890.6 -0.50 -1.42 0.006882415 0.53

0046700 // heterocycle catabolic process 890.6 -0.50 -1.42 0.007291632 0.54

0044270 // cellular nitrogen compound catabolic process 898.2 -0.50 -1.42 0.007732012 0.54

0009236 // cobalamin biosynthetic process 1097.4 -0.35 -1.28 0.007825894 0.54

Differential Abundance Analysis

Sample to Insight

Semi-automated low-throughput workflow for microbial analyses

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• QIAcube isolated DNA hands-free in ~1hr (cat no. 9001882)

• Sample Prep kits available on the QIAcube

◦ DNeasy PowerSoil

◦ DNeasy PowerLyzer PowerSoil

◦ QIAamp PowerFecal kit (cat no. 12830-50)

◦ DNeasy PowerWater

◦ DNeasy PowerPlant Pro

◦ DNeasy PowerClean Pro

◦ DNeasy PowerBiofilm

◦ DNeasy Ultraclean Microbial

◦ RNeasy PowerMicrobiome

• Easy, fast high quality DNA library prep

◦ QIAseq 1-step amplicon kit (16S) (cat no. 180412/180415)

◦ QIAseq FX DNA library kit (WGS) (cat no. 180473)

• QIAGEN Microbial Genomics Pro Suite provides user-friendly, highly accurate microbial

analysis

◦ 16S

◦ WGS

Sample to Insight

Questions

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Health & Medicine

Automated Nucleic Acid Purification from Diverse Sample types using dedicated Microbiome kits on the QIAcube