Jorgenson Test PCR
- Gregg Randolph
Template: 2 ul of 10ng/ul Zymo Mock Community or Karen Jorgenson’s Test DNAs
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | T3 | T12 | T3 | MC | MC | MC | MC | MC | MC | MC | MC | MC |
B | T4 | T13 | T4 | MC | MC | MC | MC | MC | MC | MC | MC | MC |
C | T5 | T14 | T5 | MC | MC | MC | MC | MC | MC | MC | MC | MC |
D | T6 | T15 | T6 | MC | MC | MC | MC | MC | MC | MC | MC | MC |
E | T7 | T12 | T7 | MC | MC | MC | MC | MC | MC | MC | MC | MC |
F | T8 | T13 | T8 | MC | MC | MC | MC | MC | MC | MC | MC | MC |
G | T9 | T14 | T9 | MC | MC | MC | MC | MC | MC | MC | MC | MC |
H | T10 | T15 | T10 | MC | MC | MC | MC | MC | MC | MC | MC | MC |
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 192 | 630 |
0.45 | 10M dNTPs | 192 | 95 |
0.3 | Kapa HiFi HotStart DNA Pol | 192 | 63 |
7.25 | HPLC H2O | 192 | 1522 |
11 | Total Volume | 192 | 99 |
Add 11 ul to each well of 2 hard shell, full skirt plates, Seal with bubble strips, store in refrigerator until needed labeled “PCR1”.
Add 2 ul template
Add 2 ul primers (16S0H8 and ITS0H8)
Run on Thermocycler Program GSAF36:
Temp C | Cycles | Time |
|---|---|---|
95* | 1X | 3:00* |
98 | 36X | 0:30 |
62 | 36X | 0:30 |
72 | 36X | 0:30 |
72 | 1X | 5:00 |
4 | 1X | 0:00 |
Purify samples using GSAF’s modified MagBead protocol:
Equilibrate Beads to room Temperature
Combine ITS sample with 16S
Add 24 ul (0.8 x 60 ul) of MagBeads to each well; Pipette mix up and down 10 times
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 108 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE to reach sample
Incubate at RT for 2 minutes
Place sample plate back on magnet for 2 minutes or until all wells are cleared.
Transfer 40 ul to a clean PCR plate or wells.
qPCR:
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
10 ul | KAPA SYBR FAST qPCR MM (2X) | 30 | 300 |
2 ul | Primer Premix (10X) | 30 | 60 |
4 ul | Ultra Pure Water | 30 | 120 |
16 ul | Total Volume | 30 | 480 |
Pool of 2 ul from each well then dilute 1 ul in 999 ul H2O.
Dilute 1 ul of the first 3 columns in 999 ul H2O.
Run on qPCR
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
A | T3 | T12 | T3 | Pool |
| 20 pM | 20 pM | 20 pM |
|
|
|
|
B | T4 | T13 | T4 | Pool |
| 2 pM | 2 pM | 2 pM |
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|
C | T5 | T14 | T5 | Pool |
| 0.2 pM | 0.2 pM | 0.2 pM |
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D | T6 | T15 | T6 |
|
| 0.02 pM | 0.02 pM | 0.02 pM |
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E | T7 | T12 | T7 |
|
| 0.002 pM | 0.002 pM | 0.002 pM |
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F | T8 | T13 | T8 |
|
| 0.0002 pM | 0.0002 pM | 0.0002 pM |
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G | T9 | T14 | T9 |
|
| NTC | NTC | NTC |
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H | T10 | T15 | T10 |
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| NTC | NTC | NTC |
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qPCR Results:
All of Karen’s PCRed samples amplified to above minimums for all Illumina platforms with which I (Gregg R) have dealt. The largest requirement is for the NovaSeq at 1 nM. Hers were all above 1.5 nM.
Sample Name | Detector | Task | Ct | StdDev Ct | Qty | Mean Qty | StdDev Qty |
NTC | Standards | NTC | 27.87 | 0.312 |
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|
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NTC | Standards | NTC | 28.48 | 0.312 |
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|
|
NTC | Standards | NTC | 28.34 | 0.312 |
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NTC | Standards | NTC | 28.11 | 0.312 |
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NTC | Standards | NTC | 28.64 | 0.312 |
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NTC | Standards | NTC | 28.67 | 0.312 |
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1 | Standards | Standard | 8.51 | 0.068 | 20 |
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1 | Standards | Standard | 8.64 | 0.068 | 20 |
|
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1 | Standards | Standard | 8.62 | 0.068 | 20 |
|
|
2 | Standards | Standard | 12.01 | 0.097 | 2 |
|
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2 | Standards | Standard | 12.13 | 0.097 | 2 |
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2 | Standards | Standard | 12.2 | 0.097 | 2 |
|
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3 | Standards | Standard | 15.43 | 0.193 | 2.00E-01 |
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3 | Standards | Standard | 15.56 | 0.193 | 2.00E-01 |
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3 | Standards | Standard | 15.18 | 0.193 | 2.00E-01 |
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4 | Standards | Standard | 19.04 | 0.116 | 2.00E-02 |
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4 | Standards | Standard | 19.01 | 0.116 | 2.00E-02 |
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4 | Standards | Standard | 19.22 | 0.116 | 2.00E-02 |
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5 | Standards | Standard | 23.62 | 0.742 | 2.00E-03 |
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5 | Standards | Standard | 22.57 | 0.742 | 2.00E-03 |
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6 | Standards | Standard | 25.66 | 0.021 | 2.00E-04 |
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6 | Standards | Standard | 25.7 | 0.021 | 2.00E-04 |
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6 | Standards | Standard | 25.67 | 0.021 | 2.00E-04 |
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Pool | Standards | Unknown | 7.11 | 0.089 | 54.19 | 58.08 | 3.383 |
Pool | Standards | Unknown | 6.95 | 0.089 | 60.38 | 58.08 | 3.383 |
Pool | Standards | Unknown | 6.97 | 0.089 | 59.67 | 58.08 | 3.383 |
T10_A | Standards | Unknown | 11.71 |
| 2.56 |
|
|
T10_B | Standards | Unknown | 11.51 |
| 2.92 |
|
|
T12_A | Standards | Unknown | 10.54 |
| 5.59 |
|
|
T12_B | Standards | Unknown | 8.18 |
| 26.73 |
|
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T13_A | Standards | Unknown | 9.78 |
| 9.24 |
|
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T13_B | Standards | Unknown | 9.61 |
| 10.33 |
|
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T14_A | Standards | Unknown | 10.87 |
| 4.48 |
|
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T14_B | Standards | Unknown | 11.2 |
| 3.6 |
|
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T15_A | Standards | Unknown | 7.26 |
| 49.22 |
|
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T15_B | Standards | Unknown | 7.34 |
| 46.75 |
|
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T3_A | Standards | Unknown | 9.44 |
| 11.55 |
|
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T3_B | Standards | Unknown | 9.28 |
| 12.86 |
|
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T4_A | Standards | Unknown | 11.34 |
| 3.27 |
|
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T4_B | Standards | Unknown | 9.89 |
| 8.57 |
|
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T5_A | Standards | Unknown | 12.51 |
| 1.51 |
|
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T5_B | Standards | Unknown | 10.57 |
| 5.45 |
|
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T6_A | Standards | Unknown | 11.06 |
| 3.96 |
|
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T6_B | Standards | Unknown | 10.48 |
| 5.82 |
|
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T7_A | Standards | Unknown | 9 |
| 15.5 |
|
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T7_B | Standards | Unknown | 8.83 |
| 17.38 |
|
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T8_A | Standards | Unknown | 7.88 |
| 32.49 |
|
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T8_B | Standards | Unknown | 7.75 |
| 35.57 |
|
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T9_A | Standards | Unknown | 8.6 |
| 20.2 |
|
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T9_B | Standards | Unknown | 8.63 |
| 19.83 |
|
|
Sequence on iSeq:
Pool’s average concentration from qPCR was 58 nM.
1 nM 16S Pool: 8.6 Pool + 491 ul “10 mM Tris 8.5”
Add 5 ul 1 nM Pool to 40 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX
Remove iSeq100 Cartridge v2 from large freezer #3 36 hours to 1 week before sequencing and place in refrigerator 5 with room around it for full airflow
Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.
Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100, sbsuser password: “GenomeTechnologies”.
Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.
Bioinformatics:
Checking for PhiX
module load swset/2018.05
module load gcc/7.3.0
module load usearch/10.0.240
usearch -filter_phix QcCheck1stepPcr_S1_L001_R1_001.fastq
139857 hits (3.8%)
usearch -filter_phix QcCheck1stepPcr_S1_L001_R2_001.fastq
136627 hits (3.7%)
Demultiplexing
perl /home/grandol1/BioinformaticsTools-master/parse_barcodes_pairedend.pl QcCheck1step_Demux.csv QcCheck1stepPcr_S1_L001_R1_001.fastq QcCheck1stepPcr_S1_L001_R2_001.fastq FS10000611
head parsereport_QcCheck1stepPcr_S1_L001_R1_001.fastq
Good barcode pair count: 3317211
Bad barcode pair count: 333258
Rename to original_:
mv miderrors_QcCheck1stepPcr_S1_L001_R2_001.fastq original_miderrors_QcCheck1stepPcr_S1_L001_R2_001.fastq
mv parsed_QcCheck1stepPcr_S1_L001_R1_001.fastq original_parsed_QcCheck1stepPcr_S1_L001_R1_001.fastq
mv parsed_QcCheck1stepPcr_S1_L001_R2_001.fastq original_parsed_QcCheck1stepPcr_S1_L001_R2_001.fastq
mv parsereport_QcCheck1stepPcr_S1_L001_R1_001.fastq original_parsereport_QcCheck1stepPcr_S1_L001_R1_001.fastq
Generate Report in R
Utilized this plate map and the parse report from above as input for this r-script and generated this report. The controls are Zymo Mock Community which is all bacterial and fungal DNA. “read percent” is the percent of reads a sample represented in the total number of reads produced. “read percent KJ only” is the same calculation limited to your samples.
samplename | wellposition | pair | forwardmid | reversemid | count | locus | readpercent | readpercentKJonly |
T10 | H1 | TTGGTAAGAA_CATACTAA | TTGGTAAGAA | CATACTAA | 389 | 16S | 0.0231711196118212 | 0.292241696654621 |
T10_B | H3 | CCGCGCGAA_TGCGAAGCAA | CCGCGCGAA | TGCGAAGCAA | 1437 | 16S | 0.0855961410853138 | 1.07956637041823 |
T12 | A2 | TCCTCTTGAA_CCAGAACCAA | TCCTCTTGAA | CCAGAACCAA | 2463 | 16S | 0.146710713634745 | 1.85036323614481 |
T12_B | E2 | GATGCGCA_TCGGAGCAA | GATGCGCA | TCGGAGCAA | 13188 | 16S | 0.785554564114905 | 9.90766965419318 |
T13 | B2 | ACGAGTCAA_AGACGCAA | ACGAGTCAA | AGACGCAA | 4787 | 16S | 0.285141772703825 | 3.59630077605571 |
T13_B | F2 | CTTCATCA_CCAACGCAA | CTTCATCA | CCAACGCAA | 5524 | 16S | 0.329041811659898 | 4.14998234529596 |
T14 | C2 | GGTAATGCAA_TAGATCAA | GGTAATGCAA | TAGATCAA | 1443 | 16S | 0.0859535362464216 | 1.08407395442833 |
T14_B | G2 | TCCGCTCA_ATCGCGCAA | TCCGCTCA | ATCGCGCAA | 1709 | 16S | 0.101798055055533 | 1.28391017887596 |
T15 | D2 | TCCATATCAA_AATAAGCAA | TCCATATCAA | AATAAGCAA | 16283 | 16S | 0.969910901386336 | 12.2328317394015 |
T15_B | H2 | GAGCTTCA_CCTTATCAA | GAGCTTCA | CCTTATCAA | 20964 | 16S | 1.24873869291059 | 15.7494985312789 |
T3 | A1 | TTATTCGAA_AACGCCAA | TTATTCGAA | AACGCCAA | 8083 | 16S | 0.481470847872367 | 6.07246692560233 |
T3_B | A3 | GGAATGGAA_CGGTCCAA | GGAATGGAA | CGGTCCAA | 0 | 16S | 0 | 0 |
T4 | B1 | CTTGCAGCAA_TGAAGCCAA | CTTGCAGCAA | TGAAGCCAA | 1960 | 16S | 0.116749085961875 | 1.47247744329835 |
T4_B | B3 | AGAACCGCAA_AACCGCCAA | AGAACCGCAA | AACCGCCAA | 2023 | 16S | 0.120501735153507 | 1.51980707540437 |
T5 | C1 | AATTACCA_ATCGGACCAA | AATTACCA | ATCGGACCAA | 2367 | 16S | 0.14099239105702 | 1.77824189198326 |
T5_B | C3 | ATAATCCA_CTGATCCAA | ATAATCCA | CTGATCCAA | 1370 | 16S | 0.0816052284529436 | 1.02923168230548 |
T6 | D1 | AGGCCAGAA_CTGATACCAA | AGGCCAGAA | CTGATACCAA | 592 | 16S | 0.0352629892293012 | 0.444748288996236 |
T6_B | D3 | CGAACGCA_ACCAAGCCAA | CGAACGCA | ACCAAGCCAA | 1373 | 16S | 0.0817839260334975 | 1.03148547431053 |
T7 | E1 | GCATCAGAA_TGAGAGAA | GCATCAGAA | TGAGAGAA | 8171 | 16S | 0.486712643568615 | 6.13857815775042 |
T7_B | E3 | ATAAGAGAA_TAGGAGCCAA | ATAAGAGAA | TAGGAGCCAA | 6801 | 16S | 0.405107415115671 | 5.10934647544494 |
T8 | F1 | CCGTCAAGAA_GGTAGGAA | CCGTCAAGAA | GGTAGGAA | 11775 | 16S | 0.701388003674022 | 8.84613361981534 |
T8_B | F3 | TCCATAGAA_GATACGCCAA | TCCATAGAA | GATACGCCAA | 7931 | 16S | 0.472416837124303 | 5.95827479734654 |
T9 | G1 | CTCCGAAGAA_GGCCATAA | CTCCGAAGAA | GGCCATAA | 12398 | 16S | 0.738497534569047 | 9.31417109286374 |
T9_B | G3 | ACTGACGAA_TTAACTCCAA | ACTGACGAA | TTAACTCCAA | 10431 | 16S | 0.621331487585879 | 7.83643480155361 |
T10 | H1 | TTATGCATCA_CTATCGGCCA | TTATGCATCA | CTATCGGCCA | 2408 | ITS | 0.146972925365464 | 1.79558114043264 |
T10_B | H3 | TAGACTCCA_GCTGCGTAA | TAGACTCCA | GCTGCGTAA | 1118 | ITS | 0.0682374296339654 | 0.833662672343726 |
T12 | A2 | GCTTGCCTCA_AAGTTGCTAA | GCTTGCCTCA | AAGTTGCTAA | 2641 | ITS | 0.161194142811541 | 1.96932300327351 |
T12_B | E2 | ACCTGAAC_CTGATGGA | ACCTGAAC | CTGATGGA | 11154 | ITS | 0.680787379371422 | 8.31723921942926 |
T13 | B2 | CGAGGAAGCA_TGCAACTTAA | CGAGGAAGCA | TGCAACTTAA | 6270 | ITS | 0.382691130415888 | 4.67537115885077 |
T13_B | F2 | GCGGTAAC_ACTGAATA | GCGGTAAC | ACTGAATA | 3661 | ITS | 0.223450116180633 | 2.72990969897172 |
T14 | C2 | CATCTCAGCA_CTTGCCGA | CATCTCAGCA | CTTGCCGA | 6794 | ITS | 0.414673610852559 | 5.06610393193495 |
T14_B | G2 | GACGAGAC_GTAGCATA | GACGAGAC | GTAGCATA | 2687 | ITS | 0.164001765139951 | 2.00362397190303 |
T15 | D2 | CAGGAACGCA_ACGTTCGA | CAGGAACGCA | ACGTTCGA | 17234 | ITS | 1.05188180886562 | 12.8509324643755 |
T15_B | H2 | TATTGGAC_GCCTTATA | TATTGGAC | GCCTTATA | 16781 | ITS | 1.02423283245758 | 12.5131424906977 |
T3 | A1 | GATCGTCCA_ATTCTGGAA | GATCGTCCA | ATTCTGGAA | 1558 | ITS | 0.0950929475578874 | 1.16175889401746 |
T3_B | A3 | TAGGCTAC_AATCCAGA | TAGGCTAC | AATCCAGA | 24595 | ITS | 1.50116241667923 | 18.3398331183309 |
T4 | B1 | TAACGGTAA_CTGGATGAA | TAACGGTAA | CTGGATGAA | 185 | ITS | 0.011291524581649 | 0.137949547749185 |
T4_B | B3 | ATATAGTA_GGAATAGA | ATATAGTA | GGAATAGA | 2999 | ITS | 0.183044768758732 | 2.23627401999896 |
T5 | C1 | TTCAATTA_CTTCAATAA | TTCAATTA | CTTCAATAA | 264 | ITS | 0.0161133107543532 | 0.196857733004243 |
T5_B | C3 | CCGCGTTA_AAGGAATAA | CCGCGTTA | AAGGAATAA | 6245 | ITS | 0.381165248715665 | 4.65672932807385 |
T6 | D1 | GTCAGACCA_ATAAGAACCA | GTCAGACCA | ATAAGAACCA | 3337 | ITS | 0.203674689345745 | 2.48831157210287 |
T6_B | D3 | TTGCGAAC_GATAGATAA | TTGCGAAC | GATAGATAA | 3650 | ITS | 0.222778728232535 | 2.72170729342987 |
T7 | E1 | AATGCAGGCA_GTCCTAACCA | AATGCAGGCA | GTCCTAACCA | 6262 | ITS | 0.382202848271817 | 4.66940577300215 |
T7_B | E3 | CTTGAGCCA_GCGCTATAA | CTTGAGCCA | GCGCTATAA | 4891 | ITS | 0.298523495831596 | 3.64708777319603 |
T8 | F1 | TTAATTGGCA_CTTATTACCA | TTAATTGGCA | CTTATTACCA | 19837 | ITS | 1.21075661149282 | 14.7919198848681 |
T8_B | F3 | GGAGCGCCA_GTAACCTAA | GGAGCGCCA | GTAACCTAA | 14275 | ITS | 0.871278450827242 | 10.6444853736196 |
T9 | G1 | AGAACTTGCA_CAGAGAGCCA | AGAACTTGCA | CAGAGAGCCA | 3568 | ITS | 0.217773836255804 | 2.66056208848159 |
T9_B | G3 | ATAATGCCA_ACCTAGTAA | ATAATGCCA | ACCTAGTAA | 7010 | ITS | 0.427857228742484 | 5.22716934984751 |
All samples amplified. There was one reaction failure, but the duplicate worked.