Minor revision to V4 region SSU rrna 806R gene primer greatly increases detection of SAR11 bacterioplankton

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Ethelbert Fleming
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1 The following supplements accompany the article Minor revision to V4 region SSU rrna 806R gene primer greatly increases detection of SAR11 bacterioplankton Amy Apprill*, Sean McNally, Rachel Parsons, Laura Weber *Corresponding author: Aquatic Microbial Ecology 75: (2015) SUPPLEMENTARY MATERIAL Description of contents Supplement 1 (this document) Table S1. Description of seawater sampling sites. Table S2. Percent abundance of SAR11 compared to the entire bacterioplankton community recovered using the V4 and the revised primers from coastal seawater in the Red Sea and coral reef waters in the Federated States of Micronesia. Table S3. Comparison of the change in Red Sea and Micronesian bacterioplankton communities using the original V4 primers and revised primers when SAR11 is removed from the communities. Figure S1. Description of the V4 primers 515F and 806R aligned with representative SAR11 sequences belonging to the major subgroups. Figure S2. Comparison of the major taxonomic groups of bacteria and archaea recovered from seawater samples using the V4 and revised primers when SAR11 is removed from all communities. Figure S3. Comparison of all taxonomic groups recovered from surface seawater with the V4 and revised primers when SAR11 is removed from all communities. Additional Materials and Methods. Descriptions of primers and barcodes. Supplement 2: separate.xls document at Table S4. Excluding SAR11, the percentage of major taxonomic groups represented in Red Sea and Micronesian seawater samples analyzed using the V4 and modified primers, as well as the percentage change of each group between the primer sets.

2 Table S1. Description of seawater sampling sites. Sample Depth (m) Latitude ( N) Longitude ( E) Description Red Sea, W coastal Red Sea Red Sea, W coastal Red Sea Red Sea, W coastal Red Sea Red Sea, W coastal Red Sea Red Sea, W coastal Red Sea Red Sea, W15 1 not available not available coastal Red Sea Red Sea, W coastal Red Sea Red Sea, W coastal Red Sea Red Sea, W coastal Red Sea Red Sea, W coastal Red Sea Micronesia, Kap1, 1m Kapingamarangi atoll, coral reef lagoon Micronesia, Kap1, 8m Kapingamarangi atoll, coral reef lagoon Micronesia, Kap3, 1m Kapingamarangi atoll, coral reef lagoon Micronesia, Kap5, 12m Kapingamarangi atoll, coral reef, forereef Micronesia, Kos1, 1m Kosrae Island, coral reef, nearshore Micronesia, Kos2, 10m Kosrae Island, coral reef, nearshore Micronesia, Olim1, 1m Olimario atoll, coral reef, forereef Micronesia, Nuk2, 10m Nukuoro Island, coral reef, forereef Micronesia, Poh1, 6m Pohnpei Island, coral reef, forereef

3 Table S2. Percent abundance of SAR11 compared to the entire bacterioplankton community recovered using the V4 and the revised primers from coastal seawater in the Red Sea and coral reef waters in the Federated States of Micronesia. Sample V4 region 515F/806R (SAR11/total sequences) Revised 515F/806RB (SAR11/total sequences) Red Sea, W1 0.9% (154/17,500) 17.5% (3061/17,500) Red Sea, W3 1.0% (127/17,500) 22.9% (4009/17,500) Red Sea, W10 1.5% (254/17,500) 22.0% (3844/17,500) Red Sea, W13 1.6% (272/17,500) 30.5% (5329/17,500) Red Sea, W14 1.1% (192/17,500) 23.0% (4032/17,500) Red Sea, W15 1.0% (167/17,500) 20.4% (3580/17,500) Red Sea, W16 0.3% (47/17,500) 22.1% (3869/17,500) Red Sea, W17 0.3% (56/17,500) 25.8% (4514/17,500) Red Sea, W18 0.6% (100/17,500) 24.9% (4361/17,500) Red Sea, W23 1.6% (273/17,500) 22.0% (3859/17,500) Micronesia, Kap1, 1m 2.9% (349/12,000) 27.5% (3305/12,000) Micronesia, Kap1, 8m 2.1% (255/12,000) 27.3% (3278/12,000) Micronesia, Kap3, 1m 1.7% (199/12,000) 25.1% (3006/12,000) Micronesia, Kap5, 12m 2.5% (305/12,000) 28.6% (3432/12,000) Micronesia, Kos1, 1m 3.9% (472/12,000) 24.8% (2972/12,000) Micronesia, Kos2, 10m 3.6% (429/12,000) 28.1% (3421/12,000) Micronesia, Olim1, 1m 3.8% (454/12,000) 26.0% (3124/12,000) Micronesia, Nuk2, 10m 2.6% (308/12,000) 27.8% (3338/12,000) Micronesia, Poh1, 6m 3.1% (374/12,000) 25.3% (3030/12,000)

4 Table S3. Comparison of the change in Red Sea and Micronesian bacterioplankton communities using the original V4 primers and revised primers when SAR11 is removed from the communities. Taxonomic group Average % of community with V4 region 515F/806R (std dev) Average % of community with revised 515F/806RB (std dev) Average % community change between primers (std dev) Archaea Acidobacteria OCS155 marine group other Actinobacteria NS4 marine group NS5 marine group other Bacteroidetes Chloroflexi Synechococcus Prochlorococcus other Cyanobacteria Deferribacteres Lentisphaerae Planctomycetes Rhodobacteraceae AEGEAN-169 marine group SAR116 OCS116 clade other Alphaproteobacteria OM60 (NOR5) clade SAR86 clade other Gammaproteobacteria other Deltaproteobacteria SAR324 clade (Marine group B) Betaproteobacteria other Proteobacteria Verrucomicrobia SAR406 clade (Marine group A) other Bacteria 7.64 (4.44) <0.01 (0.01) 3.32 (1.77) 0.14 (0.09) 1.50 (0.62) 1.29 (0.48) 3.79 (1.14) 0.07 (0.05) (9.40) 2.59 (2.90) 7.50 (3.36) 2.94 (1.04) 0.11 (0.08) 0.79 (0.40) 3.16 (0.72) 5.53 (2.13) 1.61 (0.40) 1.23 (0.53) 4.32 (1.33) 0.41 (0.20) (1.91) 5.87 (2.99) 1.46 (0.52) 2.16 (1.77) 0.40 (0.21) 4.85 (0.75) 1.00 (0.74) 2.93 (1.06) 8.20 (7.22) 5.89 (3.37) <0.01 (0.01) 2.89 (1.64) 0.14 (0.08) 1.33 (0.57) 1.32 (0.53) 3.44 (0.73) 0.07 (0.07) (10.56) 4.14 (5.02) 8.32 (4.56) 2.63 (1.09) 0.12 (0.10) 0.78 (0.41) 3.03 (1.12) 5.99 (2.04) 1.46 (0.38) 0.99 (0.57) 3.95 (1.16) 0.50 (0.26) 8.70 (1.82) 5.11 (1.77) 1.17 (0.47) 1.54 (1.32) 0.63 (0.74) 4.16 (0.59) 1.21 (0.92) 2.63 (1.09) 9.94 (9.33) 1.78 (1.75) <0.01 (0.01) 0.53 (0.51) 0.05 (0.04) 0.22 (0.21) 0.15 (0.12) 0.53 (0.57) 0.04 (0.03) 2.99 (2.18) 1.55 (2.30) 1.48 (2.03) 0.38 (0.22) 0.05 (0.04) 0.15 (0.12) 0.69 (0.69) 0.64 (0.41) 0.26 (0.32) 0.27 (0.14) 0.79 (0.85) 0.13 (0.11) 1.70 (1.47)* 1.90 (1.56) 0.35 (0.33) 0.62 (0.50) 0.35 (0.64) 0.82 (0.81)** 0.28 (0.26) 0.37 (0.22) 1.95 (3.42) * Significantly different between primer sets, <0.05 significance level ** Significantly different between primer sets, <0.01 significance level

5 Surface 1 Forward primer 515F Reverse complement of 806R Sequence accession and name M = A or C W = A or T; B = C, G or T; D = A, G or T GTGCCAGCMGCCGCGGTAA ATTAGAWACCCBDGTAGTCC EU805272, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC JN547474, uncultured SAR11 cluster alpha proteobacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EF573071, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC HM798795, uncultured SAR11 cluster alpha proteobacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC GU061798, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC HQ242260, uncultured SAR11 cluster alpha proteobacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC AY697876, uncultured alphaproteobacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC AF510192, Candidatus Pelagibacter ubique GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC FR684071, uncultured marine bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC HQ671959, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC JN166308, uncultured marine microorganism GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC Deep 1 Surface 4 Surface 3 Surface 2 Chesapeake- Delaware Bay LD12 freshwater HQ242227, uncultured SAR11 cluster alpha proteobacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EU804684, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EF572985, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC GU061512, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EF573447, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC AY907722, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EU802875, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EU804708, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTGGTAGTCC JN166235, uncultured marine microorganism GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTGGTAGTCC AB193884, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTGGTAGTCC GQ259234, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTGGTAGTCC HQ672557, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC HQ673752, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC GU061975, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC JN166266, uncultured marine microorganism GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC JQ678338, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC HQ673391, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC JN233172, uncultured marine bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EU703270, uncultured alpha proteobacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC HM127843, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EU801294, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC EU803976, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC JQ941818, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC HM856449, uncultured SAR11 cluster alpha proteobacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC FJ612393, uncultured bacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC FN665764, uncultured SAR11 cluster alpha proteobacterium GTGCCAGCAGCCGCGGTAA...ATTAGATACCCTCGTAGTCC Fig. S1. Description of the V4 primers 515F and 806R aligned with representative SAR11 sequences belonging to the major subgroups that were retrieved from the SILVA SSU 115 database. Basepairs containing a mismatch to the 806R primer are bolded and underlined. Mismatches occurred for most SAR11 sequences except for the Surface 4 subgroup.

6 Fig. S2. Comparison of the major taxonomic groups of bacteria and archaea recovered from seawater samples with the V4 and 515F/806RB primers when the contribution of SAR11 is removed from all communities. Samples are from coral reef waters in the Red Sea (17,500 sequences per sample) and Federated States of Micronesia (12,000 sequences per sample).

7 Fig. S3. Comparison of all taxonomic groups recovered from surface seawater using the V4 and revised primers when SAR11 is removed from all communities (A) and the same comparison using only the taxonomic groups displaying significantly differential amplification between the primer sets (B). Samples are from coral reef waters in the Red Sea (17,500 sequences per sample) and Federated States of Micronesia (12,000 sequences per sample).

8 MATERIALS AND METHODS General primer design: Forward primer: AATGATACGGCGACCACCGAGATCTACAC <i5barcode><pad><link><16sforward> Reverse primer: CAAGCAGAAGACGGCATACGAGAT <i7barcode><pad><link><16sreverse> Forward link: GT Reverse link: CC Forward pad: TATGGTAATT Reverse pad: AGTCAGTCAG 515F/806R V4 primers Forward 16S V4 primer: GTGCCAGCMGCCGCGGTAA Reverse 16S V4 primer: GGACTACHVGGGTWTCTAAT Red Sea, W1 (Delta G kcal/mol) Forward primer (i5 barcode TACGAGAC) AATGATACGGCGACCACCGAGATCTACACTACGAGACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT) Red Sea, W3 (Delta G kcal/mol) Forward primer (i5 barcode TACGAGAC) AATGATACGGCGACCACCGAGATCTACACTACGAGACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG)

9 Red Sea, W10 (Delta G kcal/mol) Forward primer (i5 barcode GATCGTGT) AATGATACGGCGACCACCGAGATCTACACGATCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AGTAGCGT) Red Sea, W13 (Delta G kcal/mol) Forward primer (i5 barcode TACGAGAC) AATGATACGGCGACCACCGAGATCTACACTACGAGACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AGTAGCGT) Red Sea, W14 (Delta G kcal/mol) Forward primer (i5 barcode TCGACGAG) AATGATACGGCGACCACCGAGATCTACACTCGACGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AGTAGCGT) Red Sea, W15 (Delta G kcal/mol) Forward primer (i5 barcode TACGAGAC) AATGATACGGCGACCACCGAGATCTACACTACGAGACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode GGAGACTA) Red Sea, W16 (Delta G kcal/mol) Forward primer (i5 barcode ACGTCTCG) AATGATACGGCGACCACCGAGATCTACACACGTCTCGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT)

10 Red Sea, W17 (Delta G kcal/mol) Forward primer (i5 barcode CTACTATA) AATGATACGGCGACCACCGAGATCTACACCTACTATATATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT) Red Sea, W18 (Delta G kcal/mol) Forward primer (i5 barcode GATCGTGT) AATGATACGGCGACCACCGAGATCTACACGATCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT) Red Sea, W23 (Delta G kcal/mol) Forward primer (i5 barcode GTCAGATA) AATGATACGGCGACCACCGAGATCTACACGTCAGATATATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AGTAGCGT) Micronesia, Kap1, 1m (Delta G kcal/mol) Forward primer (i5 barcode CGTTACTA) AATGATACGGCGACCACCGAGATCTACACCGTTACTATATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG) Micronesia, Kap1, 8m (Delta G kcal/mol) Forward primer (i5 barcode TCGACGAG) AATGATACGGCGACCACCGAGATCTACACTCGACGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG)

11 Micronesia, Kap3, 1m (Delta G kcal/mol) Forward primer (i5 barcode CTACTATA) AATGATACGGCGACCACCGAGATCTACACCTACTATATATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG) Micronesia, Kap5, 12m (Delta G kcal/mol) Forward primer (i5 barcode CTACTATA) AATGATACGGCGACCACCGAGATCTACACTACGAGACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode ACTATGTC) Micronesia, Kos1, 1m (Delta G kcal/mol) Forward primer (i5 barcode CGTTACTA) AATGATACGGCGACCACCGAGATCTACACCGTTACTATATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CGTACTCA) Micronesia, Kos2, 10m (Delta G kcal/mol) Forward primer (i5 barcode ACGTCTCG) AATGATACGGCGACCACCGAGATCTACACACGTCTCGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG) Micronesia, Olim1, 1m (Delta G kcal/mol) Forward primer (i5 barcode CTACTATA) AATGATACGGCGACCACCGAGATCTACACCTACTATATATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CGTACTCA)

12 Micronesia, Nuk2, 10m (Delta G kcal/mol) Forward primer (i5 barcode GATCGTGT) AATGATACGGCGACCACCGAGATCTACACGATCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG) Micronesia, Poh1, 6m (Delta G kcal/mol) Forward primer (i5 barcode CGTTACTA) AATGATACGGCGACCACCGAGATCTACACCGTTACTATATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG) BIOS inflow to aquaria (Delta G kcal/mol) Forward primer (i5 barcode ACTATCTG): AATGATACGGCGACCACCGAGATCTACACACTATCTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT): BIOS seawater aquaria 7, day 2 (Delta G kcal/mol) Forward primer (i5 barcode ATCGTACG): AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AGTAGCGT): CAAGCAGAAGACGGCATACGAGATAGTAGCGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT BIOS seawater aquaria 9, day 2 (Delta G kcal/mol) Forward primer (i5 barcode TACGAGAC): AATGATACGGCGACCACCGAGATCTACACTACGAGACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TCATAGAC): CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT

13 BIOS seawater aquaria 7, day 4 (Delta G kcal/mol) Forward primer (i5 barcode ACTATCTG) AATGATACGGCGACCACCGAGATCTACACACTATCTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT BIOS seawater aquaria 9, day4 (Delta G kcal/mol) Forward primer (i5 barcode TAGCGAGT) AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT BIOS seawater aquaria 7, day 6 (Delta G kcal/mol) Forward primer (i5 barcode TAGCGAGT) AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT BIOS seawater aquaria 9, day 6 (Delta G kcal/mol) Forward primer (i5 barcode TCATCGAG) AATGATACGGCGACCACCGAGATCTACACTCATCGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CGTACTCA) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT BIOS seawater aquaria 7, day 8 (Delta G kcal/mol) Forward primer (i5 barcode CTGCGTGT) AATGATACGGCGACCACCGAGATCTACACCTGCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT

14 BIOS seawater aquaria 8, day 8 (Delta G kcal/mol) Forward primer (i5 barcode CGTGAGTG) AATGATACGGCGACCACCGAGATCTACACCGTGAGTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT BIOS seawater aquaria 7, day 10 (Delta G kcal/mol) Forward primer (i5 barcode CTGCGTGT) AATGATACGGCGACCACCGAGATCTACACCTGCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT BIOS seawater aquaria 8, day 10 (Delta G kcal/mol) Forward primer (i5 barcode ACTATCTG) AATGATACGGCGACCACCGAGATCTACACACTATCTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode ACTATGTC) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT BIOS seawater aquaria 8, day 12 (Delta G kcal/mol) Forward primer (i5 barcode GGATATCT) AATGATACGGCGACCACCGAGATCTACACGGATATCTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT) CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT Read 1 primer for V4 region: TATGGTAATTGTGTGCCAGCMGCCGCGGTAA Read 2 primer for V4 region: AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT Index primer for V4 region: ATTAGAWACCCBDGTAGTCCGGCTGACTGACT

15 515F/806RB primers Forward 16S V4 primer: GTGCCAGCMGCCGCGGTAA Reverse 16S V4 primer: GGACTACNVGGGTWTCTAAT Red Sea, W1 (Delta G kcal/mol) Forward primer (i5 barcode TCATCGAG) AATGATACGGCGACCACCGAGATCTACACTCATCGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AGTAGCGT) Red Sea, W3 (Delta G kcal/mol) Forward primer (i5 barcode CGTGAGTG) AATGATACGGCGACCACCGAGATCTACACCGTGAGTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode ACTATGTC) Red Sea, W10 (Delta G kcal/mol) Forward primer (i5 barcode TAGCGAGT) AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode ACTATGTC) Red Sea, W13 (Delta G kcal/mol) Forward primer (i5 barcode ACTATCTG) AATGATACGGCGACCACCGAGATCTACACACTATCTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TAGCAGAC)

16 Red Sea, W14 Forward primer (i5 barcode ATCGTACG) AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TAGCAGAC) Red Sea, W15 (Delta G kcal/mol) Forward primer (i5 barcode GGATATCT) AATGATACGGCGACCACCGAGATCTACACGGATATCTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AGTAGCGT) Red Sea, W16 (Delta G kcal/mol) Forward primer (i5 barcode TCATCGAG) AATGATACGGCGACCACCGAGATCTACACTCATCGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode ACTATGTC) Red Sea, W17 (Delta G kcal/mol) Forward primer (i5 barcode GACACCGT) AATGATACGGCGACCACCGAGATCTACACGACACCGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode GTCGTAGT) Red Sea, W18 (Delta G kcal/mol) Forward primer (i5 barcode GGATATCT) AATGATACGGCGACCACCGAGATCTACACGGATATCTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode ACTATGTC)

17 Red Sea, W23 (Delta G kcal/mol) Forward primer (i5 barcode TAGCGAGT) AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AGTAGCGT) Micronesia, Kap1, 1m (Delta G kcal/mol) Forward primer (i5 barcode CTGCGTGT) AATGATACGGCGACCACCGAGATCTACACCTGCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CGTACTCA) Micronesia, Kap1, 8m (Delta G kcal/mol) Forward primer (i5 barcode TCATCGAG) AATGATACGGCGACCACCGAGATCTACACTCATCGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TAGCAGAC) Micronesia, Kap3, 1m (Delta G kcal/mol) Forward primer (i5 barcode CGTGAGTG) AATGATACGGCGACCACCGAGATCTACACCGTGAGTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CGTACTCA) Micronesia, Kap5, 12m (Delta G kcal/mol) Forward primer (i5 barcode ATCGTACG) AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG)

18 Micronesia, Kos1, 1m (Delta G kcal/mol) Forward primer (i5 barcode TCATCGAG) AATGATACGGCGACCACCGAGATCTACACTCATCGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT) Micronesia, Kos2, 10m (Delta G kcal/mol) Forward primer (i5 barcode GACACCGT) AATGATACGGCGACCACCGAGATCTACACGACACCGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CAGTGAGT) Micronesia, Olim1, 1m (Delta G kcal/mol) Forward primer (i5 barcode TAGCGAGT) AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TAGCAGAC) Micronesia, Nuk2, 10m (Delta G kcal/mol) Forward primer (i5 barcode CTGCGTGT) AATGATACGGCGACCACCGAGATCTACACCTGCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG) Micronesia, Poh1, 6m (Delta G kcal/mol) Forward primer (i5 barcode TCATCGAG) AATGATACGGCGACCACCGAGATCTACACTCATCGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG)

19 Inflow to aquaria (Delta G kcal/mol) Forward primer (i5 barcode CGTGAGTG): AATGATACGGCGACCACCGAGATCTACACCGTGAGTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG): BIOS seawater aquaria 7, day 2 (Delta G kcal/mol) Forward primer (i5 barcode CGTGAGTG): AATGATACGGCGACCACCGAGATCTACACCTGCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode GTCGCTCG): BIOS seawater aquaria 9, day 2 (Delta G kcal/mol) Forward primer (i5 barcode TCGACGAG): AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode GGAGACTA): BIOS seawater aquaria 7, day 4 (Delta G kcal/mol) Forward primer (i5 barcode GGATATCT) AATGATACGGCGACCACCGAGATCTACACGGATATCTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode CTACGCAG) BIOS seawater aquaria 9, day4 (Delta G kcal/mol) Forward primer (i5 barcode GACACCGT) AATGATACGGCGACCACCGAGATCTACACGACACCGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TAGCAGAC)

20 BIOS seawater aquaria 7, day 6 (Delta G kcal/mol) Forward primer (i5 barcode ACTATCTG) AATGATACGGCGACCACCGAGATCTACACACTATCTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TCATAGAC) BIOS seawater aquaria 9, day 6 (Delta G kcal/mol) Forward primer (i5 barcode TCGACGAG) AATGATACGGCGACCACCGAGATCTACACTCGACGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode AACTCTCG) BIOS seawater aquaria 7, day 8 (Delta G kcal/mol) Forward primer (i5 barcode TAGCGAGT) AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TCATAGAC) BIOS seawater aquaria 8, day 8 (Delta G kcal/mol) Forward primer (i5 barcode ATCGTACG) AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode GGAGACTA) BIOS seawater aquaria 7, day 10 (Delta G kcal/mol) Forward primer (i5 barcode TCATCGAG) AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode GGAGACTA)

21 BIOS seawater aquaria 8, day 10 (Delta G kcal/mol) Forward primer (i5 barcode CGTGAGTG) AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode GGAGACTA) BIOS seawater aquaria 8, day 12 (Delta G kcal/mol) Forward primer (i5 barcode ACTATCTG) AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode GTCGCTCG) Even mock community (Delta G kcal/mol) Forward primer (i5 barcode GATCGTGT) AATGATACGGCGACCACCGAGATCTACACGATCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA Reverse primer (i7 barcode TCGCTATA) Read 1 primer for V4 region: TATGGTAATTGTGTGCCAGCMGCCGCGGTAA Read 2 primer for V4 region: AGTCAGTCAGCCGGACTACNVGGGTWTCTAAT Index primer for V4 region: ATTAGAWACCCBNGTAGTCCGGCTGACTGACT

Natural Resource Adaptation Action Area Addressing Sea Level Rise in Estuarine Ecosystems TO: TOWN OF YANKEETOWN MAY

Natural Resource Adaptation Action Area Addressing Sea Level Rise in Estuarine Ecosystems TO: TOWN OF YANKEETOWN MAY 6 2013 University of Florida Conservation Clinic Vivek Babbar, Student Associate, J.D.