Mobio Powersoil Modified Extraction Protocol

Supplementary Material:

MoBio Powersoil Modified Extraction Protocol

To the PowerBead tubes, add the swab
Vortex to mix
Check solution C1, if it is precipitated heat to 60 C until dissolved
Add 60 mcl of solution C1 and invert several times or vortex briefly
Incubate samples in 65 C for 15 minutes
Vortex the powerbead tubes horizontally for 10 minutes at maximum speed
Centrifuge the tubes at 10,000 g for 30 seconds at room temperature
Transfer the supernatant to a clean 2 ml collection tube
Add 100 mcl of solution C2 and 100 mcl of solution C3 and vortex for 5 seconds. Incubate at 4 C for 5 minutes
Centrifuge the tubes at 10,000 g for 1 minute
Avoiding the pellet, transfer up to, but no more than, 750 mcl of supernatant into a clean 2 ml collection tube
Shake to mix solution C4 before use. Add 1200 mcl of solution C4 to the supernatant and vortex for 5 seconds
Load 675 mcl onto a spin filter and centrifuge at 10,000 g for 1 minute at room temperature. Discard flow through and add an additional 675 mcl of supernatant to the spin filter and centrifuge again at 10,000 g for 1 minute at room temperature. Load the remaining supernatant onto the spin filter and centrifuge again at 10,000 g for 1 minute at room temperature
Add 500 mcl of solution C5 and centrifuge at room temperature for 30 seconds at 10,000 g
Discard the flow through
Centrifuge again at room temperature for 1 minute at 10,000 g
Carefully place the spin filter in a clean 2 ml collection tube. Avoid splashing any solution C5 onto the spin filter
Add 50 mcl of solution C6 to the center of the white filter membrane. Allow the solution to sit on the filter for 10 minutes at 55⁰C
Centrifuge at room temperature for 30 seconds at 10,000 g
Discard the spin filter. The DNA in the tube is now ready for any downstream application. No further steps are required

Fig. S1 Operational taxonomic unit (OTU) assigned phylum taxa summary plots between healthy skin and wound swabs on the skin of hellbenders (Cryptobranchus alleganiensis), as well as the two rivers that were sampled. Numbers correspond to the individual: (01-05)-eastern hellbenders, (06-12)-Ozark hellbenders; and letters correspond to the following samples:(D)-dorsum, (F)-foot, (W)-wound, (WL)-lip wound, (WLF)-left foot wound, (WRF)-right foot wound, (WB)-belly wound. The two rivers are noted as: NFWR-North Fork of the White River, and NIAG- Niangua River

Fig. S2 PCoA comparing the bacterial communities on healthy skin swabs from eastern hellbender (Cryptobranchus alleganiensis alleganiensis) dorsum (○) and feet (●). A) displays principal coordinates calculated from an unweighted UniFrac distance matrix and B) shows principal coordinated calculated from a Bray-Curtis distance matrix.

Table S1- Swab samples collected from five eastern (Cryptobranchus alleganiensis alleganiensis) and seven Ozark hellbenders (C. a. bishopi) in Missouri. Wound and leech (Placobdella cryptobranchii) presence data also presented.

Individual / Subspecies / Age Class / Healthy Skin / Wounds
1 / Eastern / Adult / Dorsum
Right Lower Foot / Absent
2 / Eastern / Adult / Dorsum
Right Lower Foot / Absent
3 / Eastern / Adult / Dorsum
Right Lower Foot / Absent
4 / Eastern / Adult / Dorsum
Right Lower Foot / Absent
5 / Eastern / Adult / Dorsum
Right Lower Foot / Absent
6 / Ozark / Adult / Dorsum
Right Lower Foot / Lower Lip
7 / Ozark / Adult / Dorsum
Right Lower Foot / Left Rear Foot
9 / Ozark / Adult / Dorsum
Right Upper Foot / Right Rear Foot
10 / Ozark / Adult / Dorsum
Right Upper Foot / Right Rear Foot
11 / Ozark / Adult / Dorsum
Left Lower Foot / Right Rear Foot
12 / Ozark / Adult / Dorsum / Left Rear Foot
Right Rear Foot
Lower Lip
Belly

Table S2- Amplification primers for the V2 region of the bacterial 16s rRNA locus and modified TruSeq Custom Amplicon Indexes used for multiplexing and sequencing of microbiome samples in Illumina instruments. Oligonucleotide sequences © 2007-2016 Illumina, Inc. All rights reserved. Derivative works created by Illumina customers are authorized for use with Illumina instruments and products only. All other uses are strictly prohibited.

Amplification Primers
Adaptor Sequence and 16S rRNA V2 Locus Sequence
Forward Primer: / ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGAGTTTGATCCTGGCTCAG
Reverse Primer: / GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCTGCCTCCCGTAGGAGT
Dual Index Barcoding Primers
Forward Primers
i5-TS-DI-501 / AATGATACGGCGACCACCGAGATCTACACTGAACCTTACACTCTTTCCCTACACGAC
i5-TS-DI-502 / AATGATACGGCGACCACCGAGATCTACACTGCTAAGTACACTCTTTCCCTACACGAC
i5-TS-DI-503 / AATGATACGGCGACCACCGAGATCTACACTGTTCTCTACACTCTTTCCCTACACGAC
i5-TS-DI-504 / AATGATACGGCGACCACCGAGATCTACACTAAGACACACACTCTTTCCCTACACGAC
i5-TS-DI-505 / AATGATACGGCGACCACCGAGATCTACACCTAATCGAACACTCTTTCCCTACACGAC
i5-TS-DI-506 / AATGATACGGCGACCACCGAGATCTACACCTAGAACAACACTCTTTCCCTACACGAC
Reverse Primers
i7-TS-DI-701 / CAAGCAGAAGACGGCATACGAGATATCACGACGTGACTGGAGTTCAGACGTG
i7-TS-DI-702 / CAAGCAGAAGACGGCATACGAGATACAGTGGTGTGACTGGAGTTCAGACGTG
i7-TS-DI-703 / CAAGCAGAAGACGGCATACGAGATCAGATCCAGTGACTGGAGTTCAGACGTG
i7-TS-DI-704 / CAAGCAGAAGACGGCATACGAGATACAAACGGGTGACTGGAGTTCAGACGTG
i7-TS-DI-705 / CAAGCAGAAGACGGCATACGAGATACCCAGCAGTGACTGGAGTTCAGACGTG
i7-TS-DI-706 / CAAGCAGAAGACGGCATACGAGATAACCCCTCGTGACTGGAGTTCAGACGTG
i7-TS-DI-707 / CAAGCAGAAGACGGCATACGAGATCCCAACCTGTGACTGGAGTTCAGACGTG
i7-TS-DI-708 / CAAGCAGAAGACGGCATACGAGATCACCACACGTGACTGGAGTTCAGACGTG
i7-TS-DI-709 / CAAGCAGAAGACGGCATACGAGATGAAACCCAGTGACTGGAGTTCAGACGTG
i7-TS-DI-710 / CAAGCAGAAGACGGCATACGAGATTGTGACCAGTGACTGGAGTTCAGACGTG
i7-TS-DI-711 / CAAGCAGAAGACGGCATACGAGATAGGGTCAAGTGACTGGAGTTCAGACGTG
i7-TS-DI-712 / CAAGCAGAAGACGGCATACGAGATAGGAGTGGGTGACTGGAGTTCAGACGTG

Table S3 – Bacterial alpha diversity scores for the dorsum skin (D) and feet (F) of eastern hellbenders (individuals 01-05, Cryptobranchus a. alleganiensis) and the dorsum skin (D), feet (F), and wounds (W) of Ozark hellbenders (individuals 06-12, C. a. bishopi).Environmental bacterial diversity metrics are also presented for the environmental samples collected along with the salamander swabs: the North Fork of the White River (Ozark hellbenders) and the Niangua River (eastern hellbenders).

Swab / Shannon-Wiener / Chao1 / Observed Species / PD whole tree
01D / 5.93 / 780.75 / 741 / 73.66
01F / 5.18 / 624.26 / 585 / 57.50
02D / 3.11 / 335.56 / 283 / 33.18
02F / 4.60 / 829.50 / 630 / 61.62
03D / 4.37 / 439.04 / 412 / 44.29
03F / 5.75 / 1134.29 / 1114 / 92.89
04D / 7.81 / 1012.38 / 967 / 86.61
04F / 7.27 / 1168.00 / 1134 / 97.11
05D / 5.55 / 823.33 / 779 / 72.23
05F / 5.43 / 1044.63 / 764 / 71.82
06D / 2.19 / 288.25 / 256 / 31.74
06F / 5.77 / 938.63 / 828 / 75.56
06W / 4.34 / 887.61 / 789 / 67.52
07D / 4.41 / 838.00 / 805 / 72.37
07F / 4.01 / 965.93 / 930 / 80.84
07W / 6.14 / 918.39 / 882 / 74.39
09D / 3.89 / 757.00 / 714 / 65.14
09W / 6.44 / 1048.96 / 1015 / 86.53
10D / 6.45 / 701.00 / 648 / 61.07
10F / 6.81 / 984.06 / 966 / 79.89
10W / 6.52 / 959.83 / 947 / 78.72
11D / 6.94 / 712.84 / 677 / 60.66
11F / 6.99 / 851.87 / 817 / 67.53
11W / 6.86 / 958.40 / 875 / 72.77
12D / 5.41 / 992.64 / 968 / 80.22
12WB / 5.19 / 840.40 / 741 / 63.10
12WL / 5.37 / 974.60 / 949 / 78.41
12WLF / 6.69 / 986.55 / 978 / 81.58
12WRF / 4.45 / 882.00 / 844 / 70.61
NFWR / 5.48 / 908.69 / 841 / 75.92
NIAG / 5.22 / 834.11 / 749 / 70.42
Eastern Dorsum / Ozark Dorsum / Ozark Wounds
Proteobacteria-54.3%
Bacteroidetes-10.0%
Cyanobacteria-10.0%
Firmicutes-8.6%
Actinobacteria-7.1%
Unassigned-5.7%
Fusobacteria-1.4%
Spirochaetes-1.4%
Verrucomicrobia-1.4% / Proteobacteria-64.9%
Bacteroidetes-16.2%
Cyanobacteria-8.1%
Acidobacteria-3.6%
Actinobacteria-2.7%
Unassigned-1.8%
Chlorobi-0.9%
Gemmatimonadetes-0.9%
Nitrospirae-0.9% / Proteobacteria-52.0%
Unassigned-19.0%
Bacteroidetes-12.0%
Cyanobacteria-5.0%
Actinobacteria-4.0%
Verrucomicrobia-2.8%
Thermini-1.2%
Acidobacteria-0.9%
Firmicutes-0.9%
Nitrospirae-0.9%
Chlorobi-0.6%
Chloroflexi-0.6%
OD1-0.6%
Planctomycetes-0.3%

Table S4 - Phylum composition of the core bacterial microbiome of eastern hellbender (Cryptobranchus a. alleganiensis)dorsum skin, Ozark hellbender (C. a. bishopi) dorsum skin, and Ozark hellbender wounds.

Table S5- Linear Discriminant Analysis associations, LDA scores, and significance values for bacterial taxa identified within eastern and Ozark hellbender dorsum swabs. Cell color is assigned based on relative abundance value from 0 (white) to 0.1 or greater (black).

eastern hellbender swabs / Ozark hellbender swabs
OTU ID / LDA / p / 01D / 02D / 03D / 04D / 05D / 06D / 07D / 09D / 10D / 11D / 12D / Taxonomy
eastern hellbender assigned OTUs / denovo167315 / 5.14 / 0.003 / 1.9E-01 / 5.0E-01 / 6.0E-02 / 6.5E-04 / 2.7E-01 / 3.0E-04 / 2.5E-05 / 2.5E-05 / 2.5E-05 / 0 / 7.5E-05 / Cetobacterium somerae
denovo188311 / 4.66 / 0.003 / 2.5E-05 / 1.1E-02 / 3.7E-01 / 2.5E-03 / 1.0E-04 / 1.1E-03 / 2.5E-03 / 1.5E-02 / 0 / 2.5E-05 / 0 / Leptospira sp
denovo233539 / 4.37 / 0.003 / 7.7E-02 / 3.2E-02 / 7.5E-03 / 5.0E-05 / 2.9E-02 / 2.5E-05 / 1.2E-04 / 1.0E-04 / 2.5E-05 / 0 / 2.5E-05 / Bacteroides sp
denovo33271 / 4.19 / 0.003 / 7.7E-02 / 1.3E-03 / 1.3E-03 / 2.5E-05 / 6.8E-03 / 5.0E-05 / 0 / 0 / 0 / 0 / 0 / Alistipes sp
denovo242526 / 4.06 / 0.003 / 1.0E-02 / 2.1E-02 / 3.3E-03 / 7.5E-05 / 4.0E-02 / 3.6E-02 / 1.7E-04 / 9.0E-04 / 5.1E-03 / 0 / 5.0E-05 / Family Enterobacteriaceae
denovo250721 / 3.90 / 0.003 / 3.5E-02 / 4.5E-03 / 2.4E-03 / 2.5E-05 / 4.0E-03 / 0 / 3.0E-04 / 0 / 0 / 0 / 0 / Order Clostridiales
denovo250953 / 3.79 / 0.003 / 1.9E-02 / 1.0E-02 / 6.5E-03 / 5.0E-05 / 5.3E-03 / 5.0E-05 / 0 / 0 / 3.5E-04 / 0 / 0 / Clostridium XI sp
denovo162651 / 3.63 / 0.003 / 2.5E-05 / 1.5E-04 / 1.4E-03 / 1.3E-02 / 3.2E-03 / 5.0E-05 / 3.5E-04 / 5.2E-04 / 0 / 1.5E-04 / 2.5E-05 / Bacillariophyta sp
denovo13484 / 3.49 / 0.003 / 1.2E-02 / 1.7E-03 / 2.9E-03 / 7.5E-05 / 2.3E-03 / 0 / 0 / 0 / 0 / 0 / 0 / Bacteroides sp
denovo130900 / 3.35 / 0.003 / 2.0E-04 / 5.3E-03 / 2.9E-03 / 2.5E-05 / 7.3E-03 / 2.5E-05 / 0 / 0 / 2.5E-05 / 0 / 0 / Family Erysipelotrichaceae
denovo236280 / 3.32 / 0.003 / 4.0E-04 / 3.0E-04 / 3.0E-04 / 6.2E-03 / 1.3E-03 / 0 / 1.2E-03 / 2.7E-04 / 4.0E-03 / 2.3E-03 / 1.6E-03 / Phylum Proteobacteria
denovo14723 / 3.32 / 0.003 / 8.9E-03 / 9.2E-04 / 9.0E-04 / 2.5E-05 / 1.2E-03 / 0 / 0 / 0 / 0 / 0 / 0 / Order Clostridiales
denovo124611 / 3.19 / 0.003 / 2.2E-04 / 2.0E-04 / 2.7E-03 / 3.7E-03 / 8.2E-04 / 0 / 7.5E-04 / 1.7E-03 / 7.0E-04 / 8.6E-03 / 4.9E-03 / Family Comamonadaceae
denovo199312 / 3.19 / 0.003 / 1.0E-04 / 5.7E-04 / 1.1E-02 / 1.0E-04 / 1.5E-04 / 0 / 4.0E-04 / 4.2E-04 / 0 / 5.5E-04 / 5.2E-04 / Naxibacter sp
denovo143058 / 3.17 / 0.003 / 1.2E-03 / 1.2E-04 / 3.9E-03 / 7.5E-05 / 4.2E-03 / 0 / 0 / 2.5E-05 / 0 / 0 / 0 / Clostridium sp
denovo109760 / 3.09 / 0.003 / 5.5E-03 / 5.0E-05 / 2.5E-05 / 5.0E-05 / 1.7E-04 / 0 / 0 / 0 / 0 / 0 / 2.5E-05 / Clostridium sp
denovo8178 / 3.08 / 0.003 / 2.5E-05 / 1.5E-03 / 1.0E-03 / 7.2E-04 / 4.0E-03 / 1.4E-02 / 1.7E-03 / 1.6E-02 / 6.2E-04 / 0 / 2.5E-05 / Unclassified Bacteria
denovo58898 / 3.08 / 0.003 / 1.0E-04 / 2.5E-05 / 1.5E-04 / 3.8E-03 / 5.0E-05 / 0 / 3.5E-04 / 5.2E-04 / 1.3E-03 / 9.0E-04 / 1.6E-03 / Haliea sp
denovo67004 / 3.07 / 0.003 / 5.0E-05 / 1.1E-03 / 4.2E-03 / 1.8E-03 / 2.7E-04 / 0 / 5.5E-04 / 2.5E-04 / 4.2E-04 / 1.2E-03 / 8.7E-04 / Albidiferax sp
denovo157959 / 2.99 / 0.003 / 3.7E-04 / 3.5E-04 / 8.5E-04 / 2.6E-03 / 2.0E-04 / 0 / 8.0E-04 / 4.0E-04 / 2.2E-04 / 1.4E-03 / 2.0E-04 / Cellvibrio sp
denovo90520 / 2.95 / 0.003 / 5.0E-05 / 1.0E-03 / 5.0E-04 / 2.2E-03 / 4.5E-04 / 0 / 5.7E-04 / 2.0E-03 / 2.5E-04 / 1.9E-03 / 7.2E-04 / Deefgea sp
denovo255898 / 2.89 / 0.003 / 5.0E-05 / 2.2E-04 / 1.0E-04 / 2.3E-03 / 2.5E-04 / 0 / 8.2E-04 / 4.7E-04 / 6.0E-03 / 7.4E-03 / 3.5E-03 / Dechloromonas sp
denovo284767 / 2.76 / 0.003 / 8.2E-04 / 2.5E-05 / 3.7E-04 / 9.2E-04 / 5.5E-04 / 0 / 1.1E-03 / 6.2E-04 / 7.2E-04 / 1.5E-03 / 8.7E-04 / Inhella sp
denovo289545 / 2.75 / 0.003 / 2.0E-04 / 3.0E-04 / 2.3E-03 / 2.5E-05 / 2.5E-04 / 9.0E-04 / 2.7E-04 / 1.2E-04 / 1.6E-02 / 0 / 0 / Corynebacterium sp
denovo252371 / 2.74 / 0.003 / 1.0E-04 / 5.5E-04 / 2.5E-04 / 1.1E-03 / 6.0E-04 / 0 / 1.7E-04 / 5.0E-04 / 1.0E-04 / 5.0E-04 / 1.7E-04 / Undibacterium sp
denovo107842 / 2.73 / 0.003 / 7.2E-04 / 2.3E-03 / 2.7E-04 / 7.5E-05 / 4.7E-04 / 1.0E-04 / 5.0E-05 / 2.0E-04 / 0 / 1.2E-04 / 0 / Citrobacter sp
denovo90708 / 2.68 / 0.003 / 2.5E-05 / 1.0E-04 / 2.5E-05 / 1.5E-04 / 2.5E-05 / 0 / 1.0E-04 / 2.5E-05 / 6.5E-04 / 3.7E-04 / 5.0E-05 / Bacillariophyta sp
denovo234042 / 2.67 / 0.003 / 2.5E-05 / 5.0E-05 / 1.2E-04 / 1.1E-03 / 7.5E-05 / 0 / 1.5E-04 / 7.5E-05 / 1.0E-04 / 5.5E-04 / 2.7E-04 / Ilumatobacter sp
denovo15307 / 2.67 / 0.003 / 2.5E-04 / 4.0E-04 / 1.2E-04 / 1.1E-03 / 7.5E-05 / 0 / 3.6E-02 / 7.3E-03 / 2.5E-05 / 7.5E-05 / 0 / Luteolibacter sp
denovo163209 / 2.64 / 0.003 / 1.0E-04 / 1.0E-04 / 2.1E-03 / 2.0E-04 / 3.2E-04 / 2.0E-04 / 0 / 2.2E-04 / 0 / 6.5E-04 / 4.5E-04 / Acinetobacter sp
denovo187008 / 2.64 / 0.003 / 2.5E-05 / 1.9E-03 / 1.3E-03 / 7.5E-05 / 1.2E-04 / 0 / 2.9E-02 / 0 / 0 / 0 / 0 / SR1 sp
denovo68045 / 2.57 / 0.003 / 7.5E-05 / 7.5E-05 / 3.2E-04 / 7.0E-04 / 3.5E-04 / 0 / 1.0E-04 / 3.7E-04 / 0 / 1.0E-04 / 0 / Polynucleobacter sp
denovo70045 / 2.54 / 0.003 / 1.0E-04 / 7.5E-05 / 4.0E-04 / 5.2E-04 / 1.5E-04 / 0 / 2.2E-04 / 1.7E-04 / 2.0E-04 / 6.2E-04 / 5.0E-04 / Pseudomonas sp
denovo24275 / 2.52 / 0.003 / 1.2E-04 / 9.0E-04 / 1.2E-04 / 1.0E-04 / 1.0E-04 / 0 / 1.7E-04 / 3.7E-04 / 2.2E-04 / 2.5E-04 / 6.2E-04 / Acinetobacter sp
denovo174986 / 2.49 / 0.003 / 1.2E-04 / 5.0E-05 / 2.5E-05 / 4.5E-04 / 2.2E-04 / 0 / 4.0E-03 / 2.5E-03 / 3.3E-03 / 4.7E-03 / 1.6E-03 / Bacillariophyta sp
denovo173455 / 2.41 / 0.003 / 2.5E-05 / 2.5E-05 / 2.5E-05 / 3.7E-04 / 3.7E-04 / 0 / 2.5E-05 / 5.0E-05 / 5.0E-05 / 2.7E-04 / 1.2E-04 / Bacillariophyta sp
denovo111185 / 2.39 / 0.003 / 2.5E-05 / 1.5E-04 / 7.5E-05 / 2.5E-04 / 3.5E-04 / 0 / 6.5E-04 / 5.0E-04 / 1.0E-04 / 7.2E-04 / 6.0E-04 / Family Rhodobacteraceae
Ozark hellbender assigned OTUs / denovo186778 / 4.50 / 0.004 / 2.5E-04 / 4.3E-03 / 0 / 5.2E-03 / 6.0E-03 / 2.5E-05 / 7.0E-03 / 4.5E-04 / 1.4E-01 / 9.7E-02 / 1.7E-02 / Bacillariophyta sp
denovo51675 / 3.80 / 0.004 / 1.9E-03 / 0 / 1.7E-04 / 2.7E-03 / 8.7E-04 / 2.5E-05 / 1.5E-02 / 4.7E-03 / 2.5E-02 / 6.4E-03 / 3.4E-03 / Bacillariophyta sp
denovo140382 / 3.56 / 0.004 / 1.4E-03 / 0 / 1.8E-03 / 9.1E-03 / 8.9E-03 / 3.7E-04 / 3.3E-03 / 3.2E-03 / 4.1E-03 / 1.2E-02 / 6.4E-03 / Family Sphingomonadaceae
denovo269570 / 3.56 / 0.004 / 0 / 3.5E-04 / 5.0E-05 / 5.0E-03 / 7.0E-04 / 2.0E-04 / 1.0E-03 / 2.7E-04 / 1.5E-02 / 1.1E-02 / 1.6E-03 / Dechloromonas sp
denovo146716 / 3.48 / 0.004 / 5.0E-05 / 0 / 2.5E-05 / 1.7E-02 / 1.0E-03 / 5.0E-05 / 2.2E-02 / 2.1E-03 / 1.2E-04 / 6.2E-04 / 2.0E-04 / Bacillariophyta sp
denovo180930 / 3.47 / 0.004 / 2.5E-04 / 0 / 5.0E-05 / 6.6E-03 / 8.5E-04 / 2.5E-05 / 9.2E-04 / 1.4E-03 / 1.1E-02 / 4.9E-03 / 7.6E-03 / Class Betaproteobacteria
denovo217812 / 3.33 / 0.004 / 2.2E-04 / 0 / 2.0E-02 / 1.1E-03 / 1.0E-04 / 5.0E-04 / 5.7E-04 / 3.4E-03 / 3.0E-04 / 4.1E-03 / 9.2E-03 / Massilia sp
denovo130427 / 3.32 / 0.004 / 0 / 0 / 0 / 1.6E-03 / 0 / 3.0E-04 / 3.2E-04 / 3.0E-04 / 6.7E-03 / 7.2E-03 / 2.2E-03 / Dechloromonas sp
denovo230479 / 3.30 / 0.004 / 4.2E-04 / 0 / 1.1E-03 / 4.7E-03 / 2.2E-03 / 2.0E-03 / 7.7E-04 / 1.6E-03 / 2.5E-03 / 6.1E-03 / 3.2E-03 / Novosphingobium sp
denovo83286 / 3.29 / 0.004 / 8.7E-04 / 0 / 5.8E-03 / 6.6E-03 / 7.6E-03 / 1.7E-04 / 3.1E-03 / 1.1E-03 / 2.7E-03 / 4.6E-03 / 4.2E-03 / Family Sphingomonadaceae
denovo97683 / 3.18 / 0.004 / 2.2E-04 / 0 / 4.5E-04 / 5.5E-03 / 1.3E-03 / 5.2E-04 / 1.5E-03 / 9.5E-04 / 2.7E-03 / 2.8E-03 / 4.0E-03 / Order Rhizobiales
denovo208984 / 3.13 / 0.004 / 0 / 3.8E-03 / 1.7E-03 / 7.5E-05 / 4.8E-03 / 3.0E-03 / 2.7E-04 / 5.0E-05 / 8.8E-03 / 4.0E-04 / 2.0E-04 / Streptophyta sp
denovo281417 / 3.11 / 0.004 / 5.0E-05 / 0 / 0 / 1.5E-03 / 9.5E-04 / 3.5E-04 / 7.0E-04 / 8.7E-04 / 5.0E-03 / 2.5E-03 / 1.4E-03 / Order Ellin6067
denovo102654 / 3.08 / 0.004 / 0 / 0 / 0 / 5.0E-04 / 0 / 2.5E-05 / 2.2E-04 / 5.0E-05 / 6.0E-03 / 3.5E-03 / 1.5E-04 / Methylobacter sp
denovo166073 / 3.05 / 0.004 / 2.0E-04 / 0 / 0 / 3.6E-03 / 8.0E-04 / 2.7E-04 / 5.2E-04 / 3.7E-04 / 2.2E-03 / 3.6E-03 / 1.7E-03 / Propionivibrio sp
denovo257060 / 3.04 / 0.004 / 5.0E-04 / 0 / 3.8E-03 / 7.2E-04 / 1.0E-04 / 2.5E-05 / 1.5E-03 / 3.7E-04 / 5.7E-04 / 1.3E-03 / 5.4E-03 / Rheinheimera sp
denovo164203 / 3.03 / 0.004 / 3.2E-04 / 0 / 5.0E-05 / 1.1E-02 / 7.7E-04 / 2.5E-05 / 8.0E-04 / 7.2E-04 / 2.3E-03 / 2.2E-03 / 2.6E-03 / Sphingomonas sp
denovo259257 / 3.02 / 0.004 / 2.5E-05 / 0 / 0 / 1.1E-03 / 5.5E-04 / 2.2E-04 / 8.7E-04 / 2.1E-03 / 1.1E-03 / 3.6E-03 / 3.2E-04 / Hydrogenophaga sp
denovo151215 / 3.00 / 0.004 / 7.5E-05 / 0 / 1.7E-04 / 1.3E-03 / 4.7E-04 / 3.0E-04 / 2.6E-03 / 1.8E-03 / 1.5E-03 / 1.2E-03 / 7.7E-04 / Flavobacterium sp
denovo193968 / 2.98 / 0.004 / 2.5E-05 / 0 / 3.0E-04 / 1.8E-03 / 4.5E-04 / 3.2E-04 / 2.2E-04 / 2.0E-04 / 4.5E-03 / 1.6E-03 / 1.3E-03 / Derxia sp
denovo36572 / 2.97 / 0.006 / 1.2E-04 / 1.5E-04 / 4.5E-04 / 2.7E-04 / 2.5E-05 / 2.3E-03 / 1.5E-03 / 1.9E-03 / 5.5E-04 / 2.1E-03 / 7.7E-04 / Acinetobacter sp
denovo230436 / 2.94 / 0.004 / 0 / 1.0E-04 / 0 / 4.2E-04 / 0 / 3.5E-04 / 6.2E-04 / 2.5E-04 / 3.7E-04 / 3.0E-04 / 5.3E-03 / Cellvibrio sp
denovo242014 / 2.93 / 0.004 / 2.5E-04 / 7.5E-05 / 0 / 5.5E-03 / 1.4E-03 / 2.5E-05 / 3.2E-04 / 4.5E-04 / 2.3E-03 / 3.0E-03 / 6.2E-04 / Ohtaekwangia sp
denovo172006 / 2.89 / 0.004 / 1.7E-04 / 0 / 5.2E-04 / 9.7E-04 / 4.0E-04 / 3.5E-04 / 5.0E-04 / 8.7E-04 / 2.0E-04 / 2.5E-03 / 1.7E-03 / Undibacterium sp
denovo99382 / 2.88 / 0.004 / 4.7E-04 / 5.0E-05 / 0 / 2.0E-03 / 1.3E-03 / 3.2E-04 / 9.0E-04 / 5.5E-04 / 8.0E-04 / 1.6E-03 / 1.7E-03 / Rhodobacter sp
denovo277142 / 2.86 / 0.004 / 2.2E-04 / 0 / 0 / 1.0E-03 / 1.4E-03 / 5.0E-05 / 5.7E-04 / 1.1E-03 / 9.7E-04 / 2.1E-03 / 7.7E-04 / Novosphingobium sp
denovo199930 / 2.84 / 0.004 / 1.7E-04 / 0 / 2.5E-05 / 1.5E-03 / 6.0E-04 / 1.0E-04 / 6.0E-04 / 3.0E-04 / 1.7E-03 / 2.0E-03 / 7.2E-04 / Order Burkholderiales
denovo221228 / 2.83 / 0.004 / 2.2E-04 / 0 / 5.5E-03 / 6.5E-04 / 6.5E-04 / 3.0E-04 / 1.0E-04 / 1.2E-03 / 4.7E-04 / 1.7E-03 / 1.5E-03 / Family Shingomonadaceae
denovo244224 / 2.82 / 0.004 / 0 / 2.5E-05 / 0 / 5.0E-05 / 5.0E-05 / 5.0E-05 / 7.5E-05 / 5.0E-05 / 7.5E-05 / 7.5E-05 / 1.7E-04 / Acinetobacter sp
denovo30370 / 2.80 / 0.004 / 2.5E-04 / 0 / 1.2E-04 / 1.5E-03 / 1.3E-03 / 1.0E-04 / 1.3E-03 / 1.5E-04 / 1.3E-03 / 1.4E-03 / 6.5E-04 / Family Comamonadaceae
denovo69545 / 2.80 / 0.004 / 2.2E-04 / 6.5E-04 / 0 / 1.0E-04 / 5.0E-04 / 1.5E-04 / 1.0E-04 / 8.5E-04 / 1.0E-04 / 3.0E-03 / 1.5E-04 / Janthinobacterium sp
denovo235034 / 2.79 / 0.004 / 2.0E-04 / 2.5E-05 / 0 / 3.6E-03 / 1.9E-03 / 7.0E-04 / 7.5E-04 / 1.5E-04 / 6.7E-04 / 1.2E-03 / 1.2E-03 / Arcicella sp
denovo127149 / 2.79 / 0.004 / 7.5E-05 / 0 / 2.2E-04 / 2.3E-03 / 3.5E-04 / 2.5E-05 / 9.7E-04 / 6.2E-04 / 5.7E-04 / 5.2E-04 / 1.9E-03 / Order Rhizobiales
denovo161436 / 2.78 / 0.004 / 7.5E-05 / 0 / 0 / 1.5E-04 / 1.0E-04 / 5.0E-05 / 3.0E-04 / 2.2E-04 / 2.2E-04 / 2.5E-04 / 2.7E-04 / Bacillariophyta sp
denovo84139 / 2.78 / 0.004 / 5.0E-05 / 0 / 7.7E-04 / 5.0E-05 / 4.0E-04 / 5.0E-05 / 6.2E-04 / 4.5E-04 / 1.0E-04 / 1.6E-03 / 1.8E-03 / Unclassified Bacteria
denovo79134 / 2.75 / 0.004 / 5.0E-05 / 0 / 0 / 2.2E-04 / 5.0E-05 / 2.0E-04 / 1.4E-03 / 9.0E-04 / 8.0E-04 / 6.2E-04 / 2.2E-04 / Fluviicola sp
denovo7337 / 2.73 / 0.004 / 0 / 1.0E-04 / 2.2E-04 / 1.7E-04 / 0 / 6.0E-04 / 6.2E-04 / 8.0E-04 / 2.1E-03 / 1.5E-04 / 4.2E-04 / Acinetobacter sp
denovo130158 / 2.72 / 0.004 / 5.0E-05 / 0 / 3.0E-04 / 2.0E-03 / 3.5E-04 / 2.5E-05 / 1.4E-03 / 5.0E-05 / 9.0E-04 / 3.2E-04 / 1.2E-03 / Flavobacterium sp
denovo77890 / 2.72 / 0.004 / 2.7E-04 / 0 / 2.0E-04 / 1.5E-03 / 6.7E-04 / 4.2E-04 / 7.2E-04 / 5.2E-04 / 6.2E-04 / 1.0E-03 / 4.0E-04 / Family Comamonadaceae
denovo248922 / 2.71 / 0.004 / 1.0E-04 / 0 / 2.5E-04 / 1.6E-03 / 1.5E-04 / 9.7E-04 / 6.7E-04 / 3.0E-04 / 7.5E-04 / 4.5E-04 / 4.7E-04 / Nitrospira sp
denovo52138 / 2.68 / 0.004 / 5.0E-05 / 0 / 0 / 2.7E-04 / 5.0E-05 / 2.5E-05 / 5.0E-05 / 1.0E-04 / 2.2E-04 / 7.5E-05 / 1.0E-04 / Ideonella sp
denovo43731 / 2.68 / 0.004 / 1.5E-03 / 0 / 5.2E-04 / 8.2E-04 / 1.0E-03 / 2.5E-04 / 3.5E-04 / 8.5E-04 / 1.0E-03 / 1.0E-03 / 1.7E-04 / Arcobacter sp
denovo146792 / 2.66 / 0.004 / 0 / 0 / 0 / 2.5E-04 / 5.0E-05 / 3.2E-04 / 1.0E-04 / 7.5E-05 / 2.2E-03 / 7.2E-04 / 4.0E-04 / Gp6 sp
denovo265928 / 2.66 / 0.004 / 1.2E-04 / 2.7E-04 / 2.5E-05 / 5.0E-05 / 0 / 2.2E-04 / 4.2E-04 / 8.0E-04 / 2.5E-04 / 1.3E-03 / 3.5E-04 / Acinetobacter sp
denovo239828 / 2.63 / 0.004 / 0 / 0 / 0 / 1.2E-04 / 0 / 3.7E-04 / 2.5E-04 / 5.0E-05 / 1.7E-03 / 1.0E-03 / 2.5E-05 / Phylum Bacteroidetes
denovo262109 / 2.61 / 0.004 / 2.5E-05 / 0 / 0 / 7.5E-05 / 0 / 3.5E-04 / 2.7E-04 / 5.0E-05 / 8.0E-04 / 1.3E-03 / 2.5E-04 / Family Oxalobacteraceae
denovo235411 / 2.60 / 0.004 / 7.5E-05 / 0 / 0 / 3.2E-04 / 1.7E-04 / 1.7E-04 / 2.2E-04 / 1.7E-04 / 6.2E-04 / 1.2E-04 / 3.2E-04 / Class Alphaproteobacteria
denovo112415 / 2.60 / 0.004 / 5.0E-05 / 0 / 0 / 6.0E-04 / 0 / 4.0E-04 / 1.5E-04 / 7.5E-05 / 2.0E-04 / 8.5E-04 / 1.2E-03 / Gemmatimonas sp
denovo249152 / 2.59 / 0.004 / 1.2E-04 / 0 / 0 / 7.0E-04 / 0 / 6.0E-04 / 5.0E-04 / 6.5E-04 / 7.5E-04 / 3.0E-04 / 2.2E-04 / Limnohabitans sp
denovo74700 / 2.59 / 0.004 / 2.5E-05 / 0 / 0 / 3.7E-04 / 5.0E-05 / 1.7E-04 / 6.0E-04 / 8.7E-04 / 4.2E-04 / 4.5E-04 / 7.5E-05 / Fluviicola sp
denovo253315 / 2.58 / 0.004 / 2.5E-05 / 0 / 0 / 2.2E-04 / 5.0E-05 / 5.0E-05 / 2.5E-05 / 6.5E-04 / 5.0E-05 / 1.2E-04 / 7.5E-05 / Flavobacterium sp
denovo132722 / 2.57 / 0.004 / 2.5E-05 / 0 / 0 / 1.0E-03 / 1.2E-04 / 5.0E-05 / 1.5E-04 / 1.0E-04 / 5.0E-04 / 6.2E-04 / 6.2E-04 / Steroidobacter sp
denovo171832 / 2.56 / 0.004 / 0 / 0 / 0 / 5.0E-05 / 0 / 5.2E-04 / 7.5E-05 / 5.0E-05 / 4.7E-04 / 1.4E-03 / 1.0E-04 / Gp3 sp
denovo23910 / 2.56 / 0.004 / 5.0E-05 / 0 / 0 / 7.5E-05 / 0 / 2.0E-04 / 1.2E-04 / 1.0E-03 / 5.5E-04 / 7.5E-05 / 3.0E-04 / Altererythrobacter sp
denovo199264 / 2.56 / 0.004 / 1.0E-04 / 0 / 0 / 8.7E-04 / 2.2E-04 / 5.0E-05 / 5.2E-04 / 4.2E-04 / 7.2E-04 / 3.2E-04 / 1.2E-04 / Fluviicola sp
denovo205133 / 2.56 / 0.004 / 0 / 1.0E-04 / 0 / 6.2E-04 / 4.5E-04 / 3.0E-04 / 7.5E-05 / 1.5E-04 / 3.0E-04 / 4.2E-04 / 4.2E-04 / Shinella sp
denovo78360 / 2.55 / 0.004 / 0 / 0 / 0 / 9.2E-04 / 1.2E-04 / 1.2E-04 / 7.5E-05 / 2.2E-04 / 1.0E-03 / 9.5E-04 / 2.0E-04 / Propionivibrio sp
denovo223773 / 2.55 / 0.004 / 7.5E-05 / 1.0E-04 / 0 / 9.5E-04 / 1.0E-04 / 2.5E-05 / 3.0E-04 / 1.5E-04 / 7.7E-04 / 5.2E-04 / 4.7E-04 / Family Comamonadaceae
denovo222896 / 2.52 / 0.004 / 2.7E-04 / 0 / 1.7E-04 / 3.6E-03 / 8.0E-04 / 2.5E-05 / 4.5E-04 / 3.5E-04 / 1.0E-04 / 4.0E-04 / 4.5E-04 / Flavobacterium sp
denovo3050 / 2.51 / 0.004 / 2.5E-05 / 0 / 0 / 0 / 0 / 7.7E-04 / 2.5E-04 / 6.0E-04 / 7.2E-04 / 1.2E-04 / 5.0E-05 / Acinetobacter sp
denovo86383 / 2.51 / 0.004 / 0 / 1.7E-04 / 5.0E-05 / 0 / 0 / 7.5E-05 / 7.5E-05 / 2.0E-04 / 7.5E-05 / 6.7E-04 / 3.7E-04 / Delftia sp
denovo166447 / 2.50 / 0.004 / 7.5E-05 / 0 / 0 / 1.1E-03 / 0 / 1.2E-04 / 1.0E-04 / 3.5E-04 / 1.7E-04 / 8.0E-04 / 4.0E-04 / Order Rhizobiales
denovo80616 / 2.50 / 0.004 / 7.5E-05 / 0 / 1.7E-04 / 1.7E-04 / 0 / 1.2E-04 / 2.5E-05 / 4.2E-04 / 1.2E-04 / 2.5E-04 / 1.7E-04 / Chryseobacterium sp
denovo109963 / 2.49 / 0.004 / 3.0E-04 / 0 / 0 / 5.5E-04 / 1.0E-04 / 2.5E-04 / 3.2E-04 / 2.5E-04 / 2.5E-05 / 1.7E-04 / 4.5E-04 / Hydrogenophaga sp
denovo33010 / 2.49 / 0.004 / 0 / 0 / 0 / 5.7E-04 / 7.5E-05 / 3.5E-04 / 2.5E-04 / 2.5E-05 / 2.5E-04 / 7.2E-04 / 4.0E-04 / Family Xanthomonadaceae
denovo288138 / 2.48 / 0.004 / 0 / 0 / 0 / 1.1E-03 / 2.2E-04 / 3.5E-04 / 3.7E-04 / 5.0E-05 / 2.2E-04 / 6.7E-04 / 3.7E-04 / Order Sphingobacteriales
denovo154464 / 2.47 / 0.004 / 0 / 0 / 0 / 7.5E-05 / 0 / 3.2E-04 / 1.0E-04 / 5.0E-05 / 2.2E-04 / 3.5E-04 / 2.0E-04 / Class Gammaproteobacteria
denovo212193 / 2.47 / 0.004 / 1.0E-04 / 0 / 1.8E-03 / 5.0E-05 / 5.0E-05 / 2.2E-04 / 2.7E-04 / 4.2E-04 / 5.0E-05 / 6.5E-04 / 3.5E-04 / Brevundimonas sp
denovo225933 / 2.46 / 0.004 / 0 / 0 / 5.0E-05 / 2.0E-04 / 5.0E-05 / 2.5E-05 / 4.0E-04 / 2.0E-04 / 7.5E-05 / 2.0E-04 / 4.5E-04 / Order Burkholderiales
denovo214532 / 2.46 / 0.004 / 1.2E-04 / 0 / 0 / 4.5E-04 / 2.5E-05 / 5.0E-04 / 2.5E-04 / 1.0E-04 / 1.0E-04 / 3.5E-04 / 6.0E-04 / Steroidobacter sp
denovo213223 / 2.45 / 0.004 / 0 / 0 / 0 / 5.7E-04 / 0 / 2.5E-05 / 5.0E-05 / 1.0E-04 / 3.0E-04 / 8.2E-04 / 3.7E-04 / Unclassified Bacteria
denovo210300 / 2.44 / 0.004 / 2.7E-03 / 0 / 5.0E-05 / 1.0E-03 / 5.0E-04 / 2.0E-04 / 7.2E-04 / 7.5E-05 / 5.0E-05 / 1.0E-04 / 7.7E-04 / Flavobacterium sp
denovo101143 / 2.43 / 0.004 / 1.7E-04 / 1.7E-04 / 6.0E-04 / 1.2E-04 / 0 / 2.5E-04 / 4.2E-04 / 1.2E-04 / 5.0E-05 / 2.2E-04 / 6.5E-04 / Acinetobacter sp
denovo224212 / 2.43 / 0.004 / 5.0E-05 / 0 / 0 / 9.7E-04 / 5.7E-04 / 2.5E-05 / 3.7E-04 / 2.5E-05 / 1.7E-04 / 7.0E-04 / 2.2E-04 / Arenimonas sp
denovo69774 / 2.43 / 0.004 / 0 / 0 / 0 / 2.2E-04 / 5.0E-05 / 3.5E-04 / 1.0E-04 / 5.0E-05 / 4.5E-04 / 5.0E-05 / 7.5E-05 / order MND1
denovo135722 / 2.43 / 0.004 / 7.5E-05 / 0 / 4.7E-04 / 4.7E-04 / 1.2E-04 / 2.5E-05 / 5.0E-05 / 2.5E-05 / 2.0E-04 / 4.5E-04 / 6.2E-04 / Gp3 sp
denovo229873 / 2.43 / 0.004 / 0 / 0 / 0 / 1.5E-04 / 0 / 1.7E-04 / 1.2E-04 / 1.7E-04 / 3.7E-04 / 3.7E-04 / 1.2E-04 / Gp17 sp
denovo262481 / 2.41 / 0.004 / 0 / 0 / 0 / 2.5E-05 / 0 / 5.0E-05 / 7.5E-05 / 4.7E-04 / 2.5E-04 / 1.7E-04 / 2.5E-05 / Pantoea sp
denovo192386 / 2.41 / 0.004 / 0 / 0 / 0 / 0 / 0 / 2.5E-05 / 2.2E-04 / 5.0E-05 / 5.0E-05 / 1.2E-04 / 5.2E-04 / Chryseobacterium sp
denovo222842 / 2.25 / 0.004 / 0 / 1.2E-03 / 5.0E-05 / 2.5E-05 / 1.0E-04 / 2.2E-04 / 2.5E-05 / 3.0E-04 / 4.2E-04 / 2.5E-05 / 2.5E-05 / Streptophyta sp

Custom Python program, used to manipulate raw data.

#------

#Split Library Manipulation Program

#Removes any reads fewer than 100 bp in length and renames the reads with a name compatible with Qiime.

#Groups reads to a desired file

#7/15/2014

#By: Obed Hernandez-Gomez

#------

import sys

import re

import os

#def reading_seq(illumina_fasta_file.fasta)

#Extracts reads from fasta file, getting rid of quality score and sequencer data.

#Usage read_string=reading_seq(fasta_file_

#Output: a string of reads

def reading_seq(fasta_file):

with open(fasta_file) as file:

seq=''

seqs = []

for line in file:

clean_line=line.replace('\n','')

line_set = set(clean_line)

if line.startswith('@'):

seqs.append(seq)

seq = ''

if line_set > set(['G', 'C', 'T', 'A']):

x=1

elif line_set == set(['G', 'C', 'T', 'A']):

seq = clean_line

return seqs

#def write_file(read_string, sample_name)

#Applies names to each read based on name of sample of origin (name submitted) and a read specific ID (number from 1 to n, where n

#is the number of reads generated from the sample)

#Usage: write_file(sequences, sample_name)

def write_file(sequences, sample_name):

num = 1

for item in sequences:

ord = str(num)

out_file.write('>')

out_file.write(sample_name)

out_file.write('_')

out_file.write(ord)

out_file.write('\n')

out_file.write(item)

out_file.write('\n')

num += 1

return

#def del_0_len(sequences)

#groups both forward and reverse reads into one string. Will only group reads greater than 100bp

#Usage: clean_reads=clean_len(forward_reads, reverse_reads)

def clean_len(sequences):

delseq = []

for item in sequences:

if len(item) => 150:

delseq.append(item)

return delseq

#Groups reads based on map file, so that different samples can be grouped in desired comparison groups.

#Usage: read_map(map, input)

def read_map(map, sample_name, sequences):

map_features = []

x=0

y=0

for line in map:

if line.startswith('Sample'):

clean_line=line.replace('\n','')

array=(clean_line.split('\t'))

number_of_categories = array.__len__() - 1

else:

map_features.append(line.replace('\n',''))

array = map_features

category = 1

output_array=[]

while category != array.__len__():

output = array[category] + '.txt'

output_array.append(output)

category = category + 1

file_array = output_array

files = 0

while files != number_of_categories:

file_array[files] = open(output_array[files], 'a')

files = files +1

for item in map_features:

category_presence = []

if item.startswith(sample_name):

category_presence = item.split('\t')

category_presence.remove(sample_name)

for item in file_array:

index = file_array.index(item)

if category_presence[index] is '1':

num = 1

for line in sequences:

ord = str(num)

item.write('>')

item.write(sample_name)

item.write('_')

item.write(ord)

item.write('\n')

item.write(line)

item.write('\n')

num += 1

for item in file_array:

item.close()

return

#input forward reads

fastq_fileF = sys.argv[1]

#input reverse reads

fastq_fileR = sys.argv[2]

#input sample name

sample_name = sys.argv[3]

#input map file (Containing list of samples)

map_file = sys.argv[4]

#input name of output file

out_file = sys.argv[5]

map = open(map_file)

sequencesF = reading_seq(fastq_fileF)

sequences = clean_len(sequencesF)

read_map(map, sample_name, sequences)

Representative Sequences for OTUs assigned to Ozark hellbender wounds by LEfSe

>denovo107521 03D_20052

AGAGTTTGATCCTGGCTCAGTTACTAACTGGAGCCATAGTAAACCATGGAGTCTCGGTGGTTGGTAGGGTAGCGTTATCACTTGCCACACCGGAGACCGGGTTTGATTCCCGGCCAAGCACAAATGACATCACATCACAATAATTCTCCAT

>denovo119424 01D_19363

AGAGTTTGATCCTGGCTCAGGGTGAACGCTGGCGGCGTGCTTAAGACATGCAAGTCGAACGGCAGTCTTCGGACTGTAGTGGCGCACGGGTGAGTAACGCGTAACTGACCTACCCCAAAGTCGCGGATAACGGTTCGAAAGAATCGCTAAT

>denovo124611 01D_31069

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGTAACAGGTTAAGCTGACGAGTGGCGAACGGGTGAGTAATATATCGGAACGTGCCCAGTTGTGGGGGATAACTACTCGAAAGAGTGGCTA

>denovo140176 03D_107004

AGAGTTTGATCCTGGCTCAGTGGGTGAGGACTGTAAGTCCTCCAGGGGACCAGAAGCTCGCACGTCACAGTTATCGCTTACCACACCGGAGACCCAGGTTCAATTCCCAGCCAAGCACAAATTACATCACATTACAATAATTCTCCATAAT

>denovo146716 01D_147802

AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGTATGCCTTATACATGCAAGTCGTACGGGAGTTTTTAACTCAAGTGGCGGACGGGTGAGTAACACGTAAGAATTTGCCTTCAGGAGGGGGATAACAACTGGAAACGGTTGCTAATAC

>denovo160390 04D_62359

AGAGTTTGATCCTGGCTCAGCTCCTGGTCACCATGTGTGCTCTTTGGCAACCACTGTTATCTTCCTGTGCCTCAATTATTTAACTGCCTATTGGGAGCATGTGGAAGATGCATCAGGGTGGCTCTGTGTTCTATTACTATTAAATAGGAAT

>denovo174986 07D_12903

AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGTATGCTTTACACATGCAAGTCGAACGGGAGTTTTTAACTCTAGTGGCGGACGGGTGAGTAACACGTAAGAATTTGCCTTTAGGAGGGGGATAACAACTGGAAACGGGTGCTAATAC

>denovo177597 01D_30650

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGTAACAGGTCTTCGGATGCTGACGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCGATCGTGGGGGATAACGAAGCGAAAGCTT

>denovo179894 01D_166947

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGATCGGTAACGCGGGGGCAACCCTGGCGACGAGAGGCGGACGGGTGAGGAACGCTTGGGAATCTGCCCATCAGCGGGGGACAACCCGGGGAAACC

>denovo206854 07D_8306

AGAGTTTGATCCTGGCTCAGCACAAATTACATCACATCACAATAATTCTCCATAATTCACCAGAATTATTCATAATTCACCACAATTCTCCACACATTGTTCTCTCAGCGCCTCAGGACTCCTACGGGAGGCAGCAAGATCGGAAGAGCAC

>denovo208984 02D_5410

AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCATGCTTAACACATGCAAGTCGGACGGGAAGTGGTGTTTCCAGTGGCGGACGGGTGAGTAACGCGTAAGAACCTGCCCTTGGGAGGGGAACAACAACTGGAAACGGTTGCTAATAC

>denovo226124 04D_326827

AGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCTTAACACATGCAAGTCGAGGGGTATGCTTCTTCGGAAGCAGAGACCGGCGCACGGGTGCGTAACGCGTATGCAATCTACCTTTTACAGAGGGATAGCCCAGAGAAATTTGG

>denovo234736 04D_29610

AGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCATGCCTAACACATGCAAGTCGAACGAGACCTTCGGGTCTAGTGGCGCACGGGTGCGTAACGCGTGGGAACCTGCCTTTAGGTTCGGAATAACTCAGAGAAATTTGAGCTAATACC

>denovo244042 09D_14131

AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGCCCCTTTAAGAGGTGACAATCATTGATTGTCGGTTCCTATGGGGAGCGGCGGACGGCTGAGTAACGCGTGGGAATCTGCCCCAAAGTGAG

>denovo253431 03D_72163

AGAGTTTGATCCTGGCTCAGTGAGATCAATGTACTGATCAAGTTTTCGGATTGGGGAAATATCCAGAGAATCCTTAGATAACAGCGAAGGAGCCTCTGGAAACATGCAAACGCAACTTTTCCAGAATGCCTGGAGAGGAGGGACAGGGCCT

>denovo26217 07D_70282

AGAGTTTGATCCTGGCTCAGCACAAAATTACATCACATCACAATAATTCTCCAGAATTCACCAGAATTCTCCATAATTCACCAGAATTCTCCAGACATTGTTCTCTCAGCACCTTGGGACTCCTACGGGAGGCAGCAAGATCGGAAGAGCA

>denovo279129 01D_219306

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCGCGGGAGCAATCCTGGCGGCGAGTGGCGAACGGGTGAGTAATGTATCGGAACGTGCCCAGTAGTGGGGGATAGCCCGGCGAAAGC

>denovo284734 04D_15426

AGAGTTTGATCCTGGCTCAGGGGGTGAGGACTGTAAGTCCTCCAGGGGACCAGAAGCCCGCATGTTACAAGGTCCCAGAGTTTTGCCTCCTGATCAGGAGGGGCAGATACAGGTGGGTTTTCTCCTTTTAGAGAAGCTGCAGACATCACAA

>denovo33763 01D_248570

AGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGAGATGCTTTACACATGCAAGTCGTACGAGAGTTTCAAACTCAAGTGGCGGACGGGTGAGTAACACGTAAGAATTTGCCTTTAGGAGGGGGACAACAACTGGAAACGGTTGCTAATAC

>denovo36572 01D_35604

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGAGATGAGGTGCTTGCACCTTATCTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTCCGAAAGG

>denovo37779 03D_53565

AGAGTTTGATCCTGGCTCAGGGTATTAGGTTTCAGATCAGAGGTCCAGGAAAAGGTGATAGACCACCCTTTTGATGGCAACACCCTGTTCGGCCAGAAAGCTGATGACCTCCTTGAAAAGATAAAAGCCAAAACAGATACCGCCAAGGCCA

>denovo40522 05D_18276

AGAGTTTGATCCTGGCTCAGGGCCTCTCAGCAGGATGTAAATTCTCTTTTGGCCAATCAGTGTTTCAGCTGTACGTGAAAGCAATTAACTATTGCCCATAGAATAATCATTGGCTTGTGTATCTGTGTGCGTGATGGAGGAACAGCTTGTA

>denovo40873 02D_27179

AGAGTTTGATCCTGGCTCAGCACAAATTACATCACATCACAATAATTCTCCATAATTCACCAGAATTATCCAGAATTCACCAGAATTCTCCAGACAGTGTTCTCTCAGCGCCTCAGGACTCCTACGGGAGGCAGCAAGATCGGAAGAGCAC

>denovo42208 07D_27491

AGAGTTTGATCCTGGCTCAGTGTGTCAACGAAAAATCCCTAAAAATCACAAAATTGCGGGGTGCTTCAAGGCGCTTCGTTACAACTATTACTCTGCTGTGTCTGTCACAGCCCTTTTTAAATTCTCTTACTGTTTTTATCCCCAATACTCC

>denovo47610 07D_5792

AGAGTTTGATCCTGGCTCAGAATGCCAACAAAAACCCTTAAAAATTGAAAAATTGATGCCTTGCTCTACACAGGCTACCAAGCCTATGTATGTATGTGTGGGTTTTACGTTTGCTCGACCTCCGGGGAGGTGTTGGCCAACAGTCTGGAGA

>denovo70045 07D_177771

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGAGAAGAGCTTGCTCTTCGATTCAGCGGCGGACGGGTGAGTAATACCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGG

>denovo79105 01D_51152

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAAGGCCCTGCTTTTGTGGGGTGCTCGAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTGACTTTGGGATAACTTCAGGAA

>denovo84139 01D_39142

AGAGTTTGATCCTGGCTCAGATCTACCCCCTCCCCACCTATCTCCTATTGGGAGGCAGTTCAATGAATTTCTCACTCTTTCTGTGAAGAACTGTGTTATGTTACTCCTGCACCTCCCAACCATTACTCCCTGTGTCTGTCTCAGGCCTTTT

>denovo90520 01D_96956

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCTTTACACATGCAAGTCGAACGGTAACAGGGTGCTTGCACCGCTGACGAGTGGCGAACGGGTGAGTAATACATCGGAATGTACCCAGTAATGGGGGATAACGCTTCGAAAGG