Nature Metrics - Environmental Monitoring
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Wildlife protection: weak institutions
Should we allocate resources to patrolling, community payments, and/or snare removal? If so, where? Are nature reserves effective?
iDNA: Collect the collectors
21 of 25 leeches
contained mammal DNA
Metabarcoding
Bulk DNA extraction per tube Expensive and difficult
PCR amplification of 16S gene (16Smam + human blocker) (~89-90 bp amplicon for verts)
Amplicon sequencing: 218 M reads1,598 98% OTUs assigned to Vertebrata
Bulk samples of leeches (1 sample = 1 collecting session) ~40 leeches per tube with RNALater 468 samples from WWF
Resultsof16Smamprimerset404of468samplesweresuccessfullyamplifiedandsequenced
Rawdata 218,733,600 reads
Denoiseandmerge 163,584,969 reads
Qualitycontrol 139,580,727 reads
Mergeidenticalreads 4,325,333 uniqueseqs
Removechimeras 4,321,587 uniquenon-chimeraseqs
Clusterseqsat98%(CROP) 2,028 OTUs
BLASTandremovenon-targetOTUs 1,598 16S-vertOTUs
Awiderangeofmammals
BovidaeCervidaeTragulidaeSuidaeCanidaeMustelidae
UrsidaeHerpestidaeFelidaeChiropteraCercopithecidaeCricetidae
HystricidaeLeporidaeMuridaeSciuridaeSpalacidaeTupaiidae
Douglas Chesters
Taxonomic assignment via phylogenetic placement
Constrained to the topology from Bininda-Emonds et al.
(2007) Nature (multi-gene phylogeny)
BostaurusBubalusbubalis MuntiacusRusaunicolorTraguluskanchilCapricornissumatraensis/milneedwardsii
SusscrofaMartesflavigulaArctonyxcollarisUrsusthibetanusHerpestesjavanicusCatopumatemninckii
Prionailurusspp.Macacaspp.NesolagustimminsiNiviventerRhizomyspruinosusTupaiabelangeri
RusaunicolorCapricornis
sumatraensis/milneedwardsii
Ursusthibetanus
We are using leeches and 16S rRNA to search for the saola antelope
Wehaveaverylow-confidencedetectionofSaola(Pseudoryxnghetinensis)
289readsinHueSaoLaReserve2readsinQuangnamSaoLaNationalReserve
reference sequencequery OTU sequence
Carnivora
Is this nature reserve effective at protecting
wildlife?
How to demonstrate effectiveness to the
government?
Using leeches to create a wildlife performance indicator
for nature reserves
• 7520 leeches in 141 sample locations (total area 678 km2)
• mammals, birds, amphibians, reptiles
• repeated samples per ranger for occupancy correction
environmental data on topography, distance to human settlements
• yearly sampling by rangers to detect contractions and expansions in species ranges
• how to design ranger incentives?
Challenge: To protect wildlife populations
Govts,NGOs,
Park Mgmt
Park Mgmt, Rangers,
Communities
Patrolling, Snare removal, Payments for Ecological Services
iDNA + a lot of statistics
Informative signal, and cheaper than standard
censuses
Not timely, & challenges for incentive design
Bioinformatic challenges: Basic pipeline is pretty good
• PCR -> hybrid capture + shotgun? • Amplicon pipeline improvements?
• Sequence wrangling with QIIME scripts • remove primers and indices, split libraries by indices
• Denoise with github.com/lh3/bfc, usearch8 (maxee=1)
• Pair-merge with usearch8 • Chimera removal with uchime • Cluster with CROP (Hao et al. 2011. Bioinformatics 27:611–618.)
Bioinformatic challenges: Taxonomic assignment
• Sequence similarity • BLAST (+ MEGAN): Gallus gallus example • Naive Bayesian Classifier • HMMER? • Metagenomic binning programs?
• Sequence similarity + phylogenetic tree • SAP (Munch et al.): build local database for speed? • Geneious8 Sequence Classifier
• Phylogenetic placement on ML reference trees • RaxML/EPA • pplacer
• PROTAX • unpublished wrapper to call consensus taxonomy, takes into account known
species without a sequence • SEPP? TIPP?
25
100s to 1000s of wild bee species per country
Necessary for pollinating wild and crop plants
200 sites to have a 93% probability of detecting 2% annual decline in species richness and abundance.
3120bees x 200sites x 2yrs1&5 = 1.25 million bees to be identified to species
Conservation Biology, 27, 113–120 (2012)
Conservation Biology, 27, 113–120 (2012)
1.25 million bees identified to species
~£1.33 M @ < 2 minutes per bee, if ID’d visually by experts
Would this dataset hold up in court?
DNA extraction, sequencing (Hiseq 2000), assembly (SOAPdenovo-Trans), mito-annotation, protein-coding-gene extraction alignment (ClustalW2, MEGA6), and verification with reads (BWA)
...
Sample 1, Sample 2 Sample 10
Bulk bee samples
1. Reference construction 2. Morphological identification
#1
...1 5 3...
#2 3 1 1
2 0 1
...
3. Mitogenomic resequencing
Bulk DNA extraction and sequencing (Hiseq 2000), and mapping reads onto references (BWA)
48 bee mito-references
Taxonomist
Mito-scaffolds
...
#1
#2
#48
...
Biomasses
...
Sample 1 Sample 2 ... Sample 10
Bee spp.#33
1. Mitogenomic skimming
3. Mitogenomic resequencing
2. Morphological identification
DNA extraction, sequencing (Hiseq 2000), assembly (SOAPdenovo-Trans), mito-annotation, protein-coding-gene extraction alignment (ClustalW2, MEGA6), and verification with reads (BWA)
...
Sample 1, Sample 2 Sample 10
Bulk bee samples
1. Reference construction 2. Morphological identification
#1
...1 5 3...
#2 3 1 1
2 0 1
...
3. Mitogenomic resequencing
Bulk DNA extraction and sequencing (Hiseq 2000), and mapping reads onto references (BWA)
48 bee mito-references
Taxonomist
Mito-scaffolds
...
#1
#2
#48
...
Biomasses
...
Sample 1 Sample 2 ... Sample 10
Bee spp.#33
1. Mitogenomic skimming
3. Mitogenomic resequencing
2. Morphological identification
0.0
0.2
0.4
0.6
0.8
0 25 50 75 100% Coverage
Freq
uenc
ies
True Negatives
True Positives
0 10,000 16,000 bp
0.0%
14.2%
0.0%
73.1%
0.0%
0.0%
0.0%
0.0%
2.0%
0.0%
94.0%
0.0%
0.0%
0.0%
0.0%
0.0%
86.4%
0.0%
0.0%
0.0%
0.0%
0.0%
0.7%
2.4%
0.0%
4.7%
0.0%
1.1%
0.0%
0.0%
0.0%
0.0%
0.0%
0.8%
0.0%
0.0%
2.6%
0.0%
1.3%
69.1%
29.1%
29.4%
0.0%
0.0%
1.4%
0.0%
0.0%
0.0%
Andrena angustiorAndrena bicolorAndrena chrysoscelesAndrena cinerariaAndrena dorsataAndrena flavipesAndrena fulvagoAndrena haemorrhoaAndrena labiataAndrena minutulaAndrena nigroaeneaAndrena nitidaAndrena semilaevisAndrena subopacaApis melliferaBombus hortorumBombus lapidariusBombus lucorumBombus pascuorumBombus pratorumBombus sylvestrisBombus terrestrisHalictus rubicundusHalictus tumulorumHylaeus confususHylaeus dilatatusLasioglossum calceatumLasioglossum fulvicorneLasioglossum laevigatumLasioglossum lativentreLasioglossum leucopusLasioglossum leucozoniumLasioglossum malachurumLasioglossum minutissimumLasioglossum morioLasioglossum parvulumLasioglossum pauxillumLasioglossum punctatissimumLasioglossum villosulumLasioglossum xanthopusNomada fabricianaNomada flavaNomada flavoguttataNomada goodenianaNomada ruficornisOsmia bicornisSphecodes ephippiusSphecodes miniatus
Bee#speciesCodicote_4A_1
Codicote_6A_1
Codicote_8A_1
Collings_12A_1
Collings_6A_1
Crux_1A_2
Crux_6A_2
Crux_7A_3
Malham_2A_3
Tismans_3A_2
Codicote_4A_1
Codicote_6A_1
Codicote_8A_1
Collings_12A_1
Collings_6A_1
Crux_1A_2
Crux_6A_2
Crux_7A_3
Malham_2A_3
Tismans_3A_2
Andrena'angustiorAndrena'bicolor 2 1534
Andrena'chrysosceles 5 11077Andrena'cineraria 1 2 5395 44216Andrena'dorsata 1 7464Andrena'flavipes 5 14895Andrena'fulvago
Andrena'haemorrhoa 1 1 1 5 2830 7781 2648 35563Andrena'labiata
Andrena'minutula 9 2 4 1371 242 1810Andrena'nigroaenea 3 1 4 2 2 21272 1000 14213 19369 7184
Andrena'nitida 1 3 3 2 6511 18994 33721 51098Andrena'semilaevis 1 2337Andrena'subopaca 2 1
Apis'mellifera 1 1 1 1 385 10652 733 4609
Bombus'hortorum 1 2005Bombus'lapidarius 1 44769Bombus'lucorum 3002 8609
Bombus'pascuorum 1 1439Bombus'pratorum 1 9657Bombus'sylvestrisBombus'terrestris 2 2 4 8377 1078 5869
Halictus'rubicundus 2 3634Halictus'tumulorum 1 1520
Hylaeus'confususHylaeus'dilatatus
Lasioglossum'calceatum 1 10 10 2 13 15 5 5882 6522 290 3670 12161 1458Lasioglossum'fulvicorne
Lasioglossum'laevigatumLasioglossum'lativentreLasioglossum'leucopus 2 2 238 245
Lasioglossum'malachurum 10 4 867 4866 5185Lasioglossum'minutissimum 4 399 220
Lasioglossum'morioLasioglossum'parvulum 14 6 1293 184 1732Lasioglossum'pauxillum 5 2 3 1007 2217 852
Lasioglossum'punctatissimum 1Lasioglossum'villosulumLasioglossum'xanthopusLasioglossum'zonulum 1 160 181
Nomada'fabriciana 1 222Nomada'flava
Nomada'flavoguttataNomada'goodeniana 1 385
Nomada'ruficornis 1 513
Osmia'bicornis
Sphecodes'ephippius 3 1 1883 779Sphecodes'miniatus 4 2139
Bee#count#data Mitogenomic#read#numbers#per#species#and#specimen
We can detect bee species in bulk samples (93.7% detection rate)Morphological IDs Mitogenomic resequencing
11 bee species detected using morphology
10 bee species detected using mitogenomic resequencing
(we think we are correct)
Bee#speciesCodicote_4A_1
Codicote_6A_1
Codicote_8A_1
Collings_12A_1
Collings_6A_1
Crux_1A_2
Crux_6A_2
Crux_7A_3
Malham_2A_3
Tismans_3A_2
Codicote_4A_1
Codicote_6A_1
Codicote_8A_1
Collings_12A_1
Collings_6A_1
Crux_1A_2
Crux_6A_2
Crux_7A_3
Malham_2A_3
Tismans_3A_2
Andrena'angustiorAndrena'bicolor 2 1534
Andrena'chrysosceles 5 11077Andrena'cineraria 1 2 5395 44216Andrena'dorsata 1 7464Andrena'flavipes 5 14895Andrena'fulvago
Andrena'haemorrhoa 1 1 1 5 2830 7781 2648 35563Andrena'labiata
Andrena'minutula 9 2 4 1371 242 1810Andrena'nigroaenea 3 1 4 2 2 21272 1000 14213 19369 7184
Andrena'nitida 1 3 3 2 6511 18994 33721 51098Andrena'semilaevis 1 2337Andrena'subopaca 2 1
Apis'mellifera 1 1 1 1 385 10652 733 4609
Bombus'hortorum 1 2005Bombus'lapidarius 1 44769Bombus'lucorum 3002 8609
Bombus'pascuorum 1 1439Bombus'pratorum 1 9657Bombus'sylvestrisBombus'terrestris 2 2 4 8377 1078 5869
Halictus'rubicundus 2 3634Halictus'tumulorum 1 1520
Hylaeus'confususHylaeus'dilatatus
Lasioglossum'calceatum 1 10 10 2 13 15 5 5882 6522 290 3670 12161 1458Lasioglossum'fulvicorne
Lasioglossum'laevigatumLasioglossum'lativentreLasioglossum'leucopus 2 2 238 245
Lasioglossum'malachurum 10 4 867 4866 5185Lasioglossum'minutissimum 4 399 220
Lasioglossum'morioLasioglossum'parvulum 14 6 1293 184 1732Lasioglossum'pauxillum 5 2 3 1007 2217 852
Lasioglossum'punctatissimum 1Lasioglossum'villosulumLasioglossum'xanthopusLasioglossum'zonulum 1 160 181
Nomada'fabriciana 1 222Nomada'flava
Nomada'flavoguttataNomada'goodeniana 1 385
Nomada'ruficornis 1 513
Osmia'bicornis
Sphecodes'ephippius 3 1 1883 779Sphecodes'miniatus 4 2139
Bee#count#data Mitogenomic#read#numbers#per#species#and#specimen
We can detect bee species in bulk samples (93.7% detection rate)Morphological IDs Mitogenomic resequencing
Bombus lucorum
Bombus terrestris
Bombus lucorum
Bombus terrestris
Species-specific PCR confirmed Bombus lucorum in these two
samples
Workers cannot be differentiated morphologically
We can estimate bee biomasses (p=0.001, R2=24.9%), and from this, we should be able to follow population trajectories
35−1 0 1 2 3
−10
12
3
Biomass versus Read numbers
Read numbers, z−transformed, mito−nuc ratio−corrected
Biom
asse
s, z−t
rans
form
ed
Challenge: To protect pollinator populations
UK Govt, NGOs,
CitizensFarmers,UK Govt
Insecticide bans, Nature reserves, Agri-environment funds, Consumer choice
mitogenomics + a lot of statistics
Timely, cheap, and informative signal
Robust to contamination, scalable, and auditable
...
Sample 1, Sample 2 Sample 10
Bulk bee samples
1. Reference construction2. Morphological identification
#1
...
1 5 3...#2 3 1 1
2 0 1
...
3. Mitogenomic resequencing
48 bee mito-references
Mito-scaffolds
...
#1
#2
#48
...
Biomasses
...
Sample 1 Sample 2 ... Sample 10
Bee spp.#33
DNA extraction
Sequencing (Hiseq 2000)
Assembly (SOAPdenovo
-Trans)
Mitochondrial protein-coding-
gene annotation
Assembly verification through alignment and
read-mapping
Mitogenome reference
construction
Bulk DNA extraction
Sequencing (Hiseq 2000)
Read-mapping (BWA)
Bioinformatic challenges
• Improve biomass frequency estimate? • standard DNA aliquot?
• Assembly of reference species • Remove adapter contamination with trimmomatic • Denoise and pair-merge with bfc, usearch8 (maxee=1) • Assembly with IDBA, org.asm, DBG2OLC, ? • Annotation with MITOS webserver • 1 library per reference species: nuDNA
• Taxonomic/Read assignment • bwa • kraken etc. • what to do with nuDNA ref seqs, which will be non-orthologous?
• SNP calling (in bulk, so no phasing unless long-read sequencers) • ????
• Pollen metagenomics: reference genomes huge
−1 0 1 2 3
−10
12
3
Biomass versus Read numbers
Read numbers, z−transformed, mito−nuc ratio−corrected
Biom
asse
s, z−t
rans
form
ed
