宏基因組學(xué)介紹-Illumina

Metagenomics

Sequencing:IntroductionFor

Research

Use

Only.

Notforusein

diagnosticprocedures.?2020Illumina,

Inc.

Allrights

reserved.Objectives●

We

will

discuss

the

comprehensive

workflow

for

ametagenomics

sequencing

project-

What

is

metagenomics

sequencing-

Resources

for

getting

started-

Library

preparation

methods-

Sequencing-

Data

analysis●

This

is

an

overview

of

many

topics,

so

I

will

includehelpful

resources

along

the

way

for

more

in

depthinformation2What

ismetagenomicssequencing?3Metagenomics

sequencing●

Metagenomics

sequencing

allows

for

the

determination

ofthe

microbial

diversity

of

a

sample.●

We

may

be

looking

at

bacteria,

fungi,

archaea,

viruses,

orcombinations

of

these

present

in

a

sample.4What

starting

material

is

used

formetagenomics?●

We

start

with

isolated,

purified

DNA

from

our

samplesource

of

interest.mixedsample-

Sources

include:-

Soil-

Skin

swabs-

Water-

Plant

surfaces-

Etc.●

Metagenomics

samples

may

also

be

called

mixedsamples,

as

they

contain

a

mixture

of

different

species.5DNA

input●

It

is

very

important

to

have

highly

pure

DNA,

free

of

anyinhibitors

and

contaminants.●

When

possible,

use

DNA

purification

kits

developed

forsample

type.-

Or

a

method

described

orvalidated

for

sample

type●

DNA/RNA

isolation

considerations

for

Illumina

LibraryPreparation

Kits-

/bulletins/2016/05/dnarna-isolation-considerations-when-using-truseq-library-prep-kits.html6DNA

input●

Input

DNA

must

be

accurately

quantified,

using

afluorometric

method

for

quantifying

double

strandedDNA,

like

Qubit

or

PicoGreen.●

Control

samples

are

optional,

though

can

be

helpful.-

Commercialmock

communities-

Well-characterized

samples7What

metagenomics

is

not●

Another

method

commonly

used

with

mixed

samples

ismetatranscriptomics,

or

microbial

transcriptomics.●

Metatranscriptomics

is

the

sequencing

of

the

RNApresent

in

a

mixed

sample.●

More

information

on

metatranscriptomics

can

be

foundhere:-

/areas-of-interest/microbiology/microbial-sequencing-methods/microbial-transcriptomics.html8Resources

for

gettingstarted9Resources

for

gettingstarted●

We

have

several

pages

with

helpful

information

forstarting

a

metagenomics

sequencing

project-

Overview

of

Environmental

Metagenomics?

/areas-of-interest/microbiology/environmental-metagenomics.html-

Introduction

to

Shotgun

Metagenomic

Sequencing?

/areas-of-interest/microbiology/microbial-sequencing-methods/shotgun-metagenomic-sequencing.html-

Microbes

and

Metagenomics

in

Human

Health

(publication

review)?

/content/dam/illumina-marketing/documents/products/research_reviews/metagenomics_research_review.pdf10Library

preparation11Two

approaches

to

librarypreparation●

Targeted

(amplicon-based)

metagenomics-

Amplification

ofone

target

region

in

thesamples-

Needs

tobe

informative

for

identification

of

species

present●

Shotgun

(whole

genome)

metagenomics-

Library

creation

from

all

of

the

DNA

present

ina

sample12Targeted

librarypreparation●

We

will

discuss

two

methods

for

targeted

librarypreparation

today.-

The

Illumina

16S

metagenomicsdemonstrated

protocol-

The

Illumina

fungal

metagenomicsdemonstrated

protocol(ITSsequencing)●

We

will

also

discuss

how

to

customize

these

workflowsfor

different

metagenomic

target

regions.1316S

sequencing●

16S

is

a

bacterial

rRNA

gene,

commonly

used

for

targetedmetagenomics

sequencing●

The

16S

rRNA

gene

has

nine

variable

regions,

which

canbe

used

for

classificationV1V2V3V4V5V6V7

V8V9Nottoscale●

The

Illumina

16S

protocol

targets

the

v3

and

v4

variableregions/science/article/pii/S2001037015000318?viewFullText=true14Illumina

16S

metagenomics

demonstratedprotocol●

This

protocol

is

a

two-step

PCR

workflow●

Input

is

12.5

ng

of

DNA●

Can

be

completed

in

one

day●

Uses

Nextera?

XT

v2

indexing

primers,

which

allow

forpooling

up

to

384

samples

per

sequencing

runFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.1516S

locus

specific

PCR

primers●

In

the

protocol,

we

give

the

following

primer

sequences,used

for

amplifying

the

v3-v4

region

of

the

16S

rRNAgene.●

This

primer

design

includes

the

locus

specific

sequencesand

the

overhang

sequences

that

are

used

during

theindexing

PCR

step.●

This

primer

pair

can

be

ordered

from

the

oligo

synthesisvendor

of

your

choice.16Illumina

16S

metagenomics

demonstratedprotocol1716S

final

librariesuppermarkerlowermarker1μlof

adilution

of

the

final

16Slibraryrun

onaBioanalyzer

withaBioanalyzer

DNA1000kit18Customizing

targets●

The

locus

specific

sequences

in

the

first

pair

of

PCRprimers

can

be

changed

to

target

different

regions.●

The

following

primer

layout

can

be

used

for

customtargets.●

The

16S

protocol

provides

these

sequences,

along

withguidance

for

primer

design.19Illumina

16S

resources●

The

page

below

includes

link

to

the

full

librarypreparation

guide,

including

the

protocol,

along

with

anFAQ

document,

and

example

16S

data.-

/downloads/16s_metagenomic_sequencing_library_preparation.html20ITS

sequencing●

ITS

refers

to

the

internal

transcribed

spacer

regionsbetween

rRNA

genes.●

There

are

two

ITS

regions,

ITS1

and

ITS2,

both

of

whichcan

be

used

for

identification

of

fungal

species.ITS1ITS218S5.8S28SNottoscale●

The

Illumina

fungal

metagenomics

protocol

targets

ITS1./article/10.1007%2FPL0000630621Illumina

fungal

metagenomics

demonstratedprotocol●

This

workflow

is

very

similar

to

the

Illumina

16Sdemonstrated

protocol.●

The

primary

difference

is

in

the

first

round

of

PCR,

wherewe

are

amplifying

the

target

locus.●

Instead

of

one

primer

pair,

a

combination

of

8

forwardprimers

and

7

reverse

primers

are

used.22ITS

locus

specific

PCR

primers●

The

primer

overhangs

for

the

first

PCR

step

are

the

sameas

those

used

in

the

16S

protocol.23ITS

locus

specific

PCR

primers●

We

use

8

forward

and

7

reverse

locus

specific

primersupended

to

the

standard

overhangs.24Illumina

fungal

metagenomics

demonstratedprotocol25Fungal

metagenomics

final

librariesuppermarkerlowermarkerSize

(bp)1μlof

adilution

of

the

final

fungal

metagenomics

libraryrun

onaFragment

Analyzer

withanAATIHighSensitivity

Kit26Fungal

metagenomics

resources●

The

page

below

includes

link

to

the

full

workflow

guide,including

the

protocol,

and

helpful

library

preparationinformation.-

/downloads/fungal-metagenomic-sequencing-demonstrated-protocol-1000000064940.html●

The

following

links

to

the

data

sheet,

which

discusses

thefull

workflow

and

analysis

results.-

/content/dam/illumina-marketing/documents/products/appnotes/its-metagenomics-app-note-1270-2018-001-web.pdf27Two

approaches

to

librarypreparation●

Targeted

(amplicon-based)

metagenomics-

Amplification

ofone

target

region

in

thesamples-

Needs

tobe

informative

for

identification

of

species

present●

Shotgun

(whole

genome)

metagenomics-

Library

creation

from

all

of

the

DNA

present

ina

sample28Shotgun

librarypreparation●

We

have

two

workflows

for

preparation

of

shotgunmetagenomic

libraries,

these

are:-

Illumina

DNA

Prep,(M)

Tagmentation

(formerly,

the

Nextera?

DNAFlex

library

preparation)-

Nextera?

XT

DNA

library

preparation●

Both

of

these

workflows

use

a

similar

approach

to

librarypreparation,

with

some

key

differences.For

Research

UseOnly.

Not

for

use

indiagnostic

procedures.29Illumina

DNA

Prep,

(M)

Tagmentation●

Previously,

Nextera?

DNA

Flex●

Input:

1

ng

–

500

ng●

3

–

4

hours

for

complete

library

preparation●

Automation

compatible●

Uses

a

two-sided

size

selection/articles/10.1186/s12864-018-5096-9For

Research

UseOnly.

Not

for

use

indiagnostic

procedures.30Illumina

DNA

Prep,

(M)

TagmentationFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.31Illumina

DNA

Prep,

(M)

Tagmentation

finallibraries●

Final

libraries

have

average

sizes

~600

bp.uppermarkerlowermarker1μlof

aNexteraDNA

Flexlibraryrun

onaBioanalyzer

with

aBioanalyzer

HighSensitivityDNA

kitFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.32Illumina

DNA

Prep,

(M)

Tagmentationresources●

Library

preparation

support

page-

/sequencing/sequencing_kits/illumina-dna-prep.html●

Library

preparation

reference

guide-

/downloads/illumina-dna-prep-reference-guide-1000000025416.html●

Illumina

DNA

Prep,

(M)

Tagmentation

product

page-

/products/by-type/sequencing-kits/library-prep-kits/nextera-dna-flex.htmlFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.33Illumina

DNA

Prep,

(M)

Tagmentationresources●

Illumina

DNA

Prep,

(M)

Tagmentation

documentation

page-

/sequencing/sequencing_kits/illumina-dna-prep/documentation.html?langsel=/us/●

Nextera

Crude

Lysate

Protocol

for

Metagenomic

Whole-Genome

Sequencing

Studies-

/content/dam/illumina-marketing/documents/products/appnotes/nextera-dna-flex-crude-lysate-770-2018-006.pdf●

Nextera?

DNA

Flex

Library

Preparation

for

Soil

ShotgunMetagenomics

Analysis-

/documents/LibraryPrep/nextera-dna-flex-soil-metagenomics-app-note-1270-2019-002/Content/Source/Library-Prep/Nextera/DNA_Flex/Metagenomics/nextera-dna-flex-soil-app-note.htmlFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.34Nextera?

XT

DNA●

Input:

1

ng●

5.5

hours

for

complete

library

preparation●

Uses

a

one-sided

size

selectionFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.35Nextera?

XT

DNA

workflowFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.36Nextera?

XT

DNA

final

libraries●

Final

libraries

have

a

broad

distributions

and

averagesizes

can

be

variable.upperlowermarkermarkeruppermarkerlowermarker1μlof

aNexteraXT

DNAlibraryrun

onaBioanalyzer

with

aBioanalyzer

HighSensitivity

DNA

kitFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.37Nextera?

XT

DNA

resources●

Library

preparation

support

page-

/sequencing/sequencing_kits/nextera_xt_dna_kit.html●

Library

preparation

reference

guide-

/downloads/nextera_xt_sample_preparation_guide_15031942.htmlFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.38Illumina

DNA

Prep,

(M)

Tagmentation

andNextera?

XT

DNAIllumina?

DNA

Prep,Nextera?

XTTagmentationSmall

genomes,amplicons,

plasmids,metagenomicsSmall

andlargegenomes,amplicons,

plasmids,metagenomicsUsesMultiplexingInput384

libraries1ng384

libraries1ngto500

ngFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.39Sequencing40Where

to

sequence?●

Yo

u

may

have

the

sequencer

you

would

like

to

use

inyour

lab●

Yo

u

may

want

to

use

an

outside

sequencing

service

orcore

facility-

Academic

and

otherfacilities-

Some

facilities

will

do

library

preparation

and

s-

Each

core

will

have

submission

requirementsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.41Choosing

the

right

sequencer●

How

much

data

is

needed?●

How

many

libraries

willI

sequence?For

Research

UseOnly.

Not

for

use

indiagnostic

procedures.43Data

needs●

The

amount

of

data

needed

willdepend

on

the-

Library

preparation

method

used-

Source

ofsamples-

Expected

complexity

of

each

sample-

Analysis

workflow

that

will

be

used●

Published

literature

is

a

great

resource

for

data

andcoverage

recommendations.●

Technical

support

may

also

be

able

to

assist

with

dataand

sequencing

recommendations.44Sequencing●

The

workflow

for

setting

up

the

run

on

your

sequencerwilldepend

on

the

sequencer

you

willbe

using.●

We

have

resources

that

willguide

you

every

step

of

theway.●

If

you

are

submitting

your

samples

to

a

sequencingservice

provider,

they

will

perform

the

run

set

up

for

you.For

Research

UseOnly.

Not

for

use

indiagnostic

procedures.45Run

evaluation●

Shotgun

metagenomics

runs

and

amplicon-based

runswilllook

and

perform

differently.●

For

reviewing

run

performance,

we

can

use

BaseSpace?Sequence

Hub,

or

we

can

use

the

Sequencing

AnalysisViewer

(SAV)

software

offline.For

Research

UseOnly.

Not

for

use

indiagnostic

procedures.46Run

evaluation

–

primary

metrics●

When

reviewing

metagenomics

runs,

we

willwant

tofocus

on

the

following

primary

metrics:-

Percent

passing

filter

(%PF):

this

is

thenumber

of

clusters

on

theflow

cell,

or

nanowells

in

patterned

flow

cells,

that

are

giving

goodsignal

and

will

generate

data.-

Aligned

percentage:

this

indicates

theamount

of

phiX

present

in

therun.-

Q30:

Q-scoring

is

how

wemeasure

data

quality;a

Q-score

of

30

orhigher

is

considered

high

quality

data.-

Percent

base

(%base):

this

refers

to

theabundance

of

each

base

ineach

cycle

of

the

sequencing

run.47Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

libraries●

We

recommend

a

2x151

bp

read

length.●

Libraries

have

high

base

diversity.●

We

recommend

a

1%

phiX

spike-in

as

a

sequencingcontrol.For

Research

UseOnly.

Not

for

use

indiagnostic

procedures.48Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.49Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.50Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.51Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.52Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.53Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.54Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.55Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.56Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.57Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.58Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.59Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesIndex

readsInsert

read

1Insert

read

2For

Research

UseOnly.

Not

for

use

indiagnostic

procedures.60Sequencing

Illumina

DNA

Prep,

(M)Tagmentation

librariesFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.61Sequencing

targeted

libraries●

For

16S

libraries,

we

recommend

a

2x301

bp

read

length

ifusing

the

MiSeq?;

for

other

sequencers,

a

2x151

bp

readlength

can

be

used.●

For

fungal

metagenomics

libraries

we

recommend

a2x151

bp

read

length.●

16S

and

fungal

metagenomics

libraries

have

low

basediversity.●

A

higher

phiX

spike

in

percentage

is

required

to

increasethe

base

diversity

at

each

cycle

of

the

sequencing

run.●

Lowering

cluster

density

improves

sequencingperformance.For

Research

UseOnly.

Not

for

use

indiagnostic

procedures.62Sequencing

16S

libraries63Sequencing

16S

libraries64Sequencing

16S

libraries65Sequencing

16S

libraries66Sequencing

16S

libraries67Sequencing

16S

libraries68Sequencing

16S

libraries69Sequencing

16S

libraries70Sequencing

16S

libraries71Sequencing

16S

libraries72Sequencing

16S

libraries73Sequencing

16S

librariesIndex

readsInsert

read

1Insert

read

274Sequencing

16S

libraries75Resources●

Does

my

sequencing

run

look

good?-

/bulletins/2019/10/does-my-sequencing-run-look-good-.html●

How

much

PhiX

spike-in

is

recommended

whensequencing

low

diversity

libraries

on

Illumina

platforms?-

/bulletins/2017/02/how-much-phix-spike-in-is-recommended-when-sequencing-low-divers.html●

16S

metagenomics

sequencing

with

the

iSeq?

100System-

/content/dam/illumina-marketing/documents/products/appnotes/iseq100-16s-app-note-770-2018-009.pdfFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.76Resources●

Sequencing

Analysis

Viewer

software-

/sequencing/sequencing_software/sequencing_analysis_viewer_sav.html●

Recorded

Sequencing

Analysis

Viewer

(SAV)

webinar-

/training.html●

Supporting

documentation

for

sequencers-

/77Data

analysis78Data

analysis

for

metagenomics●

In

data

analysis,

we

want

to

assess

the

diversity

of

eachsample.●

We

also

want

to

classify

the

taxa

present.7916S

data

analysis

–

initiating

analysisFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8016S

data

analysis

–

initiating

analysisFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8116S

data

analysis

–

initiating

analysisFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8216S

data

analysis

–

initiating

analysisFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8316S

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8416S

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8516S

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8616S

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8716S

data

analysis

–

results8816S

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.8916S

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.9016S

data

analysis

–

results9116S

data

analysis

–

results9216S

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.9316S

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.9416S

data

analysis

–

results95Fungal

metagenomics

data

analysis

–

initiatinganalysisFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.96Fungal

metagenomics

data

analysis

–

initiatinganalysisFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.97Fungal

metagenomics

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.98Fungal

metagenomics

data

analysis

–

resultsFor

Research

UseOnly.

Not

for

use

indiagnostic

procedures.99Fungal

metagenomics

data

analys