The 11th APBON Workshop
June 26, 2019

Introduction of next-generation
biodiversity assessment using MIG-seq

陶山佳久

東北大学

Yoshihisa Suyama
Tohoku University, Japan

Introduction

Various levels of biological hierarchy to survey biodiversity

Among species

Among populations
within a species

Among individuals
within a population

Species A vs. B

Populations A vs. B

Individuals A vs. B→MIG-seq can be used for surveys of genetic differentiation of all these levels

Introduction

Whole genome sequence is not necessary to identify genetic differentiation→MIG-seq is a genome wide sequencing method
using reduced representation genomic libraryKey Technique

MIG-seq
Multiplexed ISSR Genotyping by sequencing

Suyama & Matsuki, 2015
Scientific Reports 5: 16963
doi: 10.1038/srep16963

Improved version will be available soon.

1. Brief introduction of MIG-seq

MIG-seq
・Quick (Three days)
・Simple (Two PCRs & NGS run)・Low cost (<10 USD/sample)
・>1000 of SNPs
・Low quality/quantity DNA
・A wide range of species

Numbers publications using MIG-seq

1. Brief introduction of MIG-seq

1. Brief introduction of MIG-seq
LeafMaterials
DNA extraction
DNA
Data Analysis

Population Genetics and Phylogeography

Library Construction
(PCR)
NGS
LibraryNGS Run>Millions ofSequences

・Quick(Three days)
・Simple(PCR-based)
・Economical(~10 USD / sample)

Phylogenetics

MIG-seq: Multiplexed ISSR Genotyping by sequencing
PCR-based procedure for constructing reduced representation libraries,involving de novo SNP discovering, and their genotyping using NGS
SSR
1st PCR:
Multiplexed PCR withtailed ISSR primers

Genome DNAPCR

Forward primer

Genome DNAAnchor for the 2nd PCR primers
Anchor

SSR

ISSR region

SSR

Reverse primer

Highly reduced representation libraryIndexed forward primerIndex
1st PCR product

2nd PCR:Tailed PCR withdual-indexed primersIndex
Indexed reverse primerIndexed libraryP5

Mix, size selection,quantification,and NGS run

Amplified genome region (ISSR)

P7

ca. 180–630
Index-2 read (5)Dark cycle

Read-2(17 dark + 80)Mix PCR products
into one tube

Fig. Three steps of MIG-seq procedure

Read-1 Dark cycle
(17 dark + 80)Index-1 read (9)Methods: Step #1
1st PCR: An effective PCR amplification of thousands ofgenome-wide regions from a genomeaSSR region

Genome DNA

1st PCR:
Multiplexed PCR
with tailed ISSR primers

F

Highly reduced represetation libraryb

Forward primer
Genome DNA

Anchor for the 2nd PCR primers
AnchorSSR

ISSR region

SSR

Reverse primer
2nd PCR:
Tailed PCR with common
Multiple
non-repetitive
regions from various inter-simple-sequence repeat (ISSR) are amplifiedand indexedprimersfrom genomic DNA by multiplexed PCR with universal tailed-ISSR primers.

Index-tagged libraryMethods: Step #1
1st PCR

Tailed ISSR primers set

Table 1. Sequences of MIG-seq primer set-1 for the 1st PCR
Name
Sequences (5!–3!)
Forward primers:(Tail + anchor: CTG) + SSR + anchor(ACT)4TG-f
CGCTCTTCCGATCTCTGACTACTACTACTTG(CTA)4TG-f
CGCTCTTCCGATCTCTGCTACTACTACTATG(TTG)4AC-f
CGCTCTTCCGATCTCTGTTGTTGTTGTTGAC(GTT)4CC-f
CGCTCTTCCGATCTCTGGTTGTTGTTGTTCC(GTT)4TC-f
CGCTCTTCCGATCTCTGGTTGTTGTTGTTTC(GTG)4AC-fCGCTCTTCCGATCTCTGGTGGTGGTGGTGAC(GT)6TC-f
CGCTCTTCCGATCTCTGGTGTGTGTGTGTTC(TG)6AC-f
CGCTCTTCCGATCTCTGTGTGTGTGTGTGAC
Reverse primers:(Tail + anchor: GAC) + SSR + anchor(ACT)4TG-r
TGCTCTTCCGATCTGACACTACTACTACTTG(CTA)4TG-r
TGCTCTTCCGATCTGACCTACTACTACTATG(TTG)4AC-r
TGCTCTTCCGATCTGACTTGTTGTTGTTGAC(GTT)4CC-r
TGCTCTTCCGATCTGACGTTGTTGTTGTTCC(GTT)4TC-r
TGCTCTTCCGATCTGACGTTGTTGTTGTTTC(GTG)4AC-r
TGCTCTTCCGATCTGACGTGGTGGTGGTGAC(GT)6TC-r
TGCTCTTCCGATCTGACGTGTGTGTGTGTTC(TG)6AC-r
TGCTCTTCCGATCTGACTGTGTGTGTGTGAC
Underlined and boldface nucleotides denote tail and anchor sequences, respectively. The difference
between forward and reverse primer sets is only in their tail sequences.

Methods: Step #1
1st PCR

Calophyllum inophyllum

1st**changed frompublished version

Methods: Step #2
2nd PCR (Tailed PCR)
This step enables the addition of complementary sequences for the binding sites of Illumina
sequencing flow cell and index (barcode) for each sample to the 1st PCR products using
indexed primers.
Indexed forward primer
Index
1st PCR productIndex
Indexed reverse primer

New version of the 2nd PCR primers
Index (5 bases)
ForwardAATGATACGGCGACCACCGAGATCTACACxxxxxACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG(index)

Reverse(index)

CAAGCAGAAGACGGCATACGAGATxxxxxxxxxGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC

Index(9 bases)Methods: Step #2
2nd PCR (Tailed PCR)

Methods: Steps #3-1: Mix, Size selection, and Quantification
1st PCR

2nd PCR(Multiplexed Tailed PCR)

(Tailed PCR + Index)

Pool,Size selection(350–800 bp)→→(AMPure XP, Beckman Coulter)

2nd PCR Primer (70)29533

1st PCR Primer (31)32 12 3 1414 3 12 2

3Read 1924

2nd PCR Primer (70)

1st PCR Primer (31)Index 2 read

34

Read 2Construction of the MIG-seq library

Index 1 read

Methods: Step #3-2
NGS Run

MiSeq Reagent kit v3 (150 cycle, Illumina)

MiSeq Sequencer (Illumina)

Amount of data from 96–192 samples of astandard species using MiSeq
No. of raw reads:

ca. 50〜60 million

No. of reads/sample:~600,000 reads

No. of SNP markers:~5000 SNPs or more

Methods: Data analysis

pyRAD, and so on.

Data analysis with Stacks（Catchen et al 2011）

Time and cost
DNA extraction（PCR quality）
1st PCR
96–192 samples(even more)
2nd PCR
adding index foreach sample

Pool,
Size select,
Quantify

Calculation is based on
run with MiSeq platformandMiSeq Reagent kit v3(150 cycle, Illumina)~384 samples (or more)(without genome info.)
Time： 3 days
Library construction: 1 day
NGS run: 1 day
Data analysis: 1 day

Cost：~1,400 US$(4~ US$/sample)

Multiplexed DNA Barcoding

KHM-7375
THA-3183
KHM-7395
80
KHM-7382
79
MMR-1479
22
SUL-0339
49
KHM-7204
KHM-7236
THA-3621
45
KHM-7203
KHM-7420
20
THA-2146
KHM-0404
2
KHM-0514
KHM-4084
99
KHM-7235
94 THA-1258
KHM-4289
KHM-1175
KHM-2301
THA-317162 KHM-1050
KHM-4370
KHM-6996
100
THA-2466
44100 THA-3534
KHM-7290
98 THA-1011
25
LAO-1045
38
VNM-2612
LAO-0628
28LAO-0197
77
69
85
VNM-1014
100
LAO-0465
Anchor for the 2nd PCR primers VNM-3365
VNM-3288
95
THA-3313
100 KHM-6732
94
KHM-7509
100
LAO-0940
Specificprimer region
Specific primer region
VNM-2824
95
94 VNM-5713
VNM-3458
100
22 VNM-5297
KHM-3013
KHM-6176
53 KHM-2429
13
KHM-6061
33
THA-2491
VNM-3380
THA-229466
MMR-0277
100 THA-5094
MMR-0027
MMR-0084
21
THA-2913
2VNM-5301
2034
THA-1301
4287 SWK-0013
SWK-3573
92
SWK-1322
96
SWK-0489
92SWK-2073
52
THA-2520
THA-3891
stMMR-1270
34
VNM-0463
VNM-0760
6100 MMR-1577
MMR-0042
9
100 THA-1582
19
THA-2840
LAO-0063
KH
2 61
37
VNM-3300
VNM-332054
LAO-0501
LAO-1161
35
VNM-0658
95 VNM-6788
VNM-6277
LAO-0595
VNM-3277
VNM-3879
100 VNM-3318
KHM-0990
VNM-3826
THA-1044
LAO-0031
ex21
KHM-1360
Ind
Amplified genome regions
SWK-0493
100
LAO-1044
99
THA-5105
KHM-6892
THA-3055
98
KHM-7491
Dark cycle
Dark cycle
Read-1
Read-2
LAO-0120
Index-1 read (9)84 SWK-2063(3 dark + 247)
98 (3 dark85+ 247)
SWK-3461
35 SWK-1553
98
VNM-3016
VNM-2985
VNM-5893
31
VNM-1140
VNM-4630
100 LAO-0986
VNM-4740
THA-4739
THA-4459
VNM-2794
VNM-5943
KHM-5566
100 VNM-5934
VNM-2856
KHM-5482100
KHM-3291
VNM-2185
THA-4613
32
100 THA-4725
92 MMR-0163
MMR-0258
VNM-6892
2899
VNM-0851
50 VNM-3677
VNM-2502
38
42 VNM-2367
VNM-2363
VNM-1637
VNM-5599
KHM-0534
67KHM-0210
100
KHM-1391
18A simple and economical protocol to detect sequences of ITS and several cpDNAregions (psbA-trnH, rbcL, trnLUAAintron and matK) together using one run of NGS.
Primers for multiplexed DNA barcoding a) 1st PCRcpDNA

nDNA

Forward primer

Multiplexed PCR with
tailed specific primers

 Genome DNAMultiplex PCR
psbA"trnH$

trnLUAAintron$
rbcLa$

Reverse primer

matK$

ITS1&2$

Multiplexed Amplicon libraryb) 2nd PCR

Indexed forward primer

rbcLa$psbA"trnH$

trnLUAAintron$

Tailed PCR withindexed primers

ITS1&2$

Target region

matK$

Index

1 PCR product

Index

Indexed reverse primerIndexed libraryc) NGS runP5Mix, size selection,quantification,and NGS runIndex-2 read (5)d) Data analysis~250 bp from both ends of each regionP7

ca. 500–850

Mix PCR products
into one tube

Illumina MiSeq Reagent Nano Kit v2 (500 cycles)=ca. 500 USD for 384 samples(< 2 USD/sample)

exIndbuilding lociAlignment

Phylogenetics

MIG-seq and Multiplexed DNA barcoding
An efficient combination for the next-generation biodiversity assessment

Phylogenetics

Phylogeography

Conservation Genetics

Take-home message

MIG-seq and Multiplexed DNA Barcoding
New tools for biodiversity assessment
Three independent genetic data based on
1) Genome-wide SNPs
2) cpDNA genes (or mtDNA, ex. CO1)
3) ITS, etc.
can be easily detected
MIG-seq can cover:Multi-barcoding:family, genus, species, population, individual
family, genus, species, population, individualAdvantages
・Applicable to a wide range of DNA quality/quantity
・Quick (three days + one–three days)
・Simple (two PCR steps and NGS run)・Economical (< 2000 USD / >384 samples)MIG-seq Analysis Package
Supported by grants from the Project of the NARO Bio-oriented
Technology Research Advancement Institution (Research
program on development of innovative technology)

Materials

MIG-seq Data
ATGAAGGTAAATGTGAAGATATTGGTGAATAATGGAGTGAAAATAGGGACCATTTTAGGCCCAACAAACACACCAGAGATDNA extraction
DNA
IK296..
V1395..

Library Construction(PCR)

V0672..
V0262..
V0504..
V0673..
V4268..
V4493..

V3339..
V0884..
V0981..

MY187..
T1922..
T2979..
MY453..

T5055..

SWK2548

V5802..
V5741..
V5742..
V0301..
V0967..
V2504..
V5697..
IS0988.
V4429..
V0593..
IS0112.
V3007..
V5559..
V3844..
T2687..
T3582..
T4309..
T4637..
V4912..
M087...
V3571..
V5639..
V5729..
V0352..
V0357..
V0453..
V0982..
V2788..
V3575..
V3669..
V5476..
V5614..
V0020..
V0069..
V0325..
V0060..
V3994..
V1429..
V3944..
V1879..
V2721..
V3596..
V4854..
V4864..
C1802..
C3125..
C4408..
T0429..
T1153..
T0663..
T1906..
T3842..

NGS
Library

T4235..
V0210..
V0801..
V1223..
T3509..
T4277..

V3859..

Identification

IS0784.
SWK1290

V4688..
V2372..
V2477..
V3032..
V1157..
V1468..
V2720..
V2905..
C0010..
C0060..
C0325..
C0324..
C6016..
C6531..
C0459..
C2630..
C0334..
C1812..
C6408..
C5503..
C2842..
C3981..
MY423..
C7319..
V5419..
V1303..

NGS Run>Millions ofSequences

C0583..
C1022..
T1823..
T4175..
T4891..
T4909..
T5304..
T530402
MY104..
T5357..
T1920..
C6756..
C6906..
C7302..
C7587..
C7583..
C7621..
C7470..
V5388..
T3932..
V4614..
T1314..
T4285..
T4386..
T4360..
T0201..
T0244..
C1444..
C4582..

V3860..

MIG-seq Data Analysis System

MIG-seq Database

C6302..
IS0095.
IS0324.
IS0354.
IS0677.
IS0703.
IK738..
SWK1137
SWK1625
SWK2540
SWK3429
SWK3424
SWK1166
SWK1188
SWK1115
SWK1284
SWK2242
SWK3053
SWK3426
SWK3469
IK635..
IS0224.
IS0792.
SWK0718
SWK1213
IJ1310.
IJ1395.
IS1052.
IS0781.
IK341..
M007...
M134...
IS0151.
M106...
M179...
C1995..
C6058..
C2979..
C5849..
C3941..
T1694..
T2410..
T5113..
C7637..

V5612..
V5753..

C6229..

MIG-seq
Species Identification System

Summary: Features of MIG-seq

Advantages
・Applicable to a wide range of DNA quality/quantity
・Quick (three days)
・Simple (two PCR steps and NGS run)・Economical (~10 US$/sample)
Disadvantages
・Inapplicable to >10000 markers
・SNPs from limited regions
Recommended studies
・Population genetics, phylogeography and phylogeny
・Identification of clones, hybrids, breeding varieties, andspecies
・Next-generation biodiversity assessment

Acknowledgements
Methods
Yu Matsuki
Ayumi Matsuo
Shun Hirota
Chica Mitsuyuki
Yoshihiro Tsunamoto
Mitsuhiko Sato
New Caledonia
Yuji Isagi
Kotomi Fujita
Gildas Gâteblé
Rimpei Nagaoka
SE Asia
Tetsukazu Yahara
Shuichiro Tagane
Hironori Toyama
Akiyo Naiki
Etsuko Moritsuka

Funding
Environment Research and Technology
Development Fund (4-1605, 4-1902) ofthe Environmental Restoration andConservation Agency of Japan
JSPS KAKENHI Grant Numbers
JP16H02553, JP18H04011, JP1802469,
NARO Bio-oriented Technology Research
Advancement Institution (Researchprogram on development of innovativetechnology), Grant Number 30031C