Deep amplicon sequencing for detection of mixed phytoplasma ...

AARHUS
UNIVERSITET
Deep amplicon sequencing for
detection of mixed phytoplasma
infections in plants
Nicoletta Contaldo ‐ Alessandro Canel ‐ Samanta Paltrinieri ‐ Assunta
Bertaccini ‐ Mogens Nicolaisen
University of Bologna – Italy & Aarhus University – Denmark

• Phytoplasmas exist as populations of
individuals within plants or insects
Sequence variation within populations
• Mutations over time
• Multiple infections of the same plant
• Vectors feeding on plants infected with
different phytoplasmas

• Phytoplasmas
– are not yet cultured in vitro to obtain single clones
– are usually identified by sequencing or PCR/RFLP analyses
from whole population
– some genotypes may be present in low titer

• Verify the deep amplicon sequencing for
phytoplasma population studies
• Study population structures of phytoplasmas
by analysing sequence variation within single
plants

Deep amplicon sequencing
• Using 454 pyrosequencing to generate large amounts of
sequence reads from single amplicons
– Reads from individual amplicons can be regarded as representatives
of individual phytoplasma cells
– Semi‐quantitative
PCR
Pyrosequencing TGATGTGCATTATTGTGAT
Sanger sequencing
Pyrosequencing TGATGTGCATTATTGTGCT

• field collected grapevine samples
• nested PCR/RFLP analyses with group specific
primers BN or FD phytoplasma
infection (single infection)

RFLP ANALYSES ON 16R758f/ B6 AMPLICONS
MW 46 51 56 53 63 64 68 71 59 33 43 66 54
16R 758f / B6 Taq I

Sequencing approach
• Use primers designed to amplify 5’ end of 16S
– Tags included to allow pooling of samples

TAGGED PRIMERS FOR GENERATING PYROSEQUENCING
SAMPLES
AMPLICONS POOLED IN EQUIMOLAR AMOUNTS AND RUN ON
AN AGAROSE GEL
PURIFIED EXPECTED BAND FROM GEL
SEQUENCED ON A GS FLX PLATE AT EUROFINS MWG
Tag-sorted sequences were quality filtered using
CLOTU software at the Bioportal webportal
(http://www.bioportal.uio.no/)
2-10,000 reads obtained from each sample

• Discard bad‐quality sequences
• Cluster sequences using CDHIT at 99% identity
• BLAST each cluster against Q‐Bank or
GenBank to identify clusters at 16Sr group or
’Ca. Phytoplasma’ level

• field collected grapevine samples
• nested PCR/RFLP analyses with group specific
primers mixed phytoplasma infection

Nested‐PCR results of selected
samples
Stolbur (‘bois noir’ - 16SrXII-A)
‘Flavescence dorée’ (16SrV-C & 16SrV-D)
identified after RFLP with TruI and TaqI

TAGGED PRIMERS FOR GENERATING PYROSEQUENCING
SAMPLES
AMPLICONS POOLED IN EQUIMOLAR AMOUNTS AND RUN ON
AN AGAROSE GEL
PURIFIED EXPECTED BAND FROM GEL
SEQUENCED ON A GS FLX PLATE AT EUROFINS MWG
Tag-sorted sequences were quality filtered using
CLOTU software at the Bioportal webportal
(http://www.bioportal.uio.no/)
2-10,000 reads obtained from each sample

• Discard bad‐quality sequences
• Cluster sequences using CDHIT at 99% identity
• BLAST each cluster against Q‐Bank or
GenBank to identify clusters at 16Sr group or
’Ca. Phytoplasma’ level

Cluster0|GZ41KDL02B6N95|487|T_TCAGTCGCGA|FPY(11-30)|RPY(247-260)|rTN|GPB|D_1_|216
Cluster41|GZ41KDL02B6F11|484|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
4
Cluster28|GZ41KDL02BQZCJ|484|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
0
Cluster16|GZ41KDL02BYPR2|482|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
Cluster21|GZ41KDL02BT5LT|489|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
Cluster38|GZ41KDL02CFG1F|496|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
10
0
Cluster11|GZ41KDL02CE5CR|487|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
Cluster18|GZ41KDL02B9B0P|486|T_TCAGTCGCGA|FPY(11-30)|RPY(247-260)|rTN|GPB|D_1_|216
5
0
Cluster47|GZ41KDL02CBM1C|533|T_TCAGTCGCGA|FPY(11-30)|RPY(250-263)|rTN|GPB|D_1_|219
0
Cluster1|GZ41KDL02CAUB2|479|T_TCAGTCGCGA|FPY(11-30)|RPY(244-257)|rTN|GPB|D_1_|213
13
Cluster49|GZ41KDL02B0I44|489|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
Cluster22|GZ41KDL02BYF0G|491|T_TCAGTCGCGA|FPY(11-30)|RPY(247-260)|rTN|GPB|D_1_|216
Cluster33|GZ41KDL02CD43H|486|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215 31
Cluster7|GZ41KDL02B2OVA|481|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
Cluster40|GZ41KDL02BZELF|483|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
41
Cluster14|GZ41KDL02B4R86|485|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
Cluster44|GZ41KDL02CLGXN|490|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
Cluster50|GZ41KDL02CFXDN|491|T_TCAGTCGCGA|FPY(11-30)|RPY(246-259)|rTN|GPB|D_1_|215
26
22
2
11
4
42
Cluster42|GZ41KDL02B935N|484|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
71
Cluster39|GZ41KDL02BY9ST|480|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster8|GZ41KDL02BV884|482|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster53|GZ41KDL02BUGMY|505|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster46|GZ41KDL02CCOYE|487|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster51|GZ41KDL02BS9YC|486|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster4|GZ41KDL02BUTL5|482|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster23|GZ41KDL02CKISW|487|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster10|GZ41KDL02B1E86|483|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster19|GZ41KDL02B1728|493|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
47
15
31
21
23
16
94
90
Cluster15|GZ41KDL02CHE2M|488|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster45|GZ41KDL02B9B18|485|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster36|GZ41KDL02B3I75|481|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster12|GZ41KDL02BT8QY|487|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster6|GZ41KDL02B0O1U|481|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster34|GZ41KDL02BSWO7|482|T_TCAGTCGCGA|FPY(11-30)|RPY(244-257)|rTN|GPB|D_1_|213
32
Cluster25|GZ41KDL02CIAVP|484|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
45
Cluster9|GZ41KDL02BTLBU|486|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
4
Cluster5|GZ41KDL02B5XF3|478|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212
Cluster13|GZ41KDL02BYUZ0|484|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster20|GZ41KDL02BZLGR|481|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
50
Cluster17|GZ41KDL02BZRY8|491|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster2|GZ41KDL02BTWYC|485|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster24|GZ41KDL02B606B|484|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster3|GZ41KDL02BZ7KW|484|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster48|GZ41KDL02B366M|482|T_TCAGTCGCGA|FPY(11-30)|RPY(245-258)|rTN|GPB|D_1_|214
Cluster26|GZ41KDL02CD1ZA|482|T_TCAGTCGCGA|FPY(11-30)|RPY(243-256)|rTN|GPB|D_1_|212 68 15
11
1100
10 37
45
80
100
0.01
Sample 112 sequences
16SrV‐C/D
16SrX‐B
16SrXII‐A

Alignment of representative
16SrXII‐A
16SrV‐C/D
16SrX‐B
sequences in sample 112

• the population structure of the mixed samples
was more complex than initially thought
• 3 different 16Sr groups were recovered
• Within 16Sr groups, sequence variation was
found

• deep amplicon sequencing allow to analyze
population structures (single or mixed
phytoplasma infection confirmation)
• and to obtain relative quantities????
• However……

….the strength of pyrosequencing is
also its weakness
• PCR errors in cluding chimeras
• Sequencing accuracy is 99.5% (1 error in 200 bases)
• Contamination
• In contrast to Sanger sequencing, these errors
may be detected as strain variations, HOWEVER,
if several sequences have the same error this
increases the likelihood of a ’real’ polymorphism

Mixed phytoplasma infection when disease is in
endemic phase
Single phytoplasma infection in epidemic phase
Further confirmation on other plants than grapevine
are in progress…
Thank you!