Freshwater Aquarium Microbiome - Revision history

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 . [https://www.youtube.com/watch?v=womKfikWlxM Illumina Sequencing Technology - Illumina YouTube Video]
 ==Page History==
 This page was created by Sara L Simonson in the spring of 2021

@@ Line 41: / Line 41: @@
 . Savage, D. C. [[Media:32_300.pdf|Microbial ecology of the gastrointestinal tract.]] Ann Rev Microbiol. '''1977'''. ''31''. 107-133.
-. Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. [[Media:pone.0039971.pdf|Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water.]] ''Public Library of Science One.'' '''2012'''. ''7''.
+.
-.
+. Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. [[Media:pone.0039971.pdf|Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water.]] ''Public Library of Science One.'' '''2012'''. ''7''.
-.
+. Patin, N. V.; Pratte, Z. A.; Regensburger, M.; Hall, E.; Gilde, K.; Dove, A. D. M.; Stewart, F. J. [[Media:AEM.00179-18.pdf|Microbiome Dynamics in a Large Artificial Seawater Aquarium. Applied and Environmental Microbiology.]] 2018. 84 (10).
 . [[Media:HB-2266-002_HB_DNY_PowerWater_Sterivex_0519_WW%20(2).pdf|DNEASY PowerWater Sterivex Kit Handbook]]

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 . Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.
+. [https://www.youtube.com/watch?v=womKfikWlxM Illumina Sequencing Technology - Illumina YouTube Video]
 ==Page History==
 This page was created by Sara L Simonson in the spring of 2021

@@ Line 47: / Line 47: @@
 .
-. [[file:///C:/Users/saras/Downloads/HB-2266-002_HB_DNY_PowerWater_Sterivex_0519_WW%20(2).pdf|DNEASY PowerWater Sterivex Kit Handbook]]
+. [[Media:HB-2266-002_HB_DNY_PowerWater_Sterivex_0519_WW%20(2).pdf|DNEASY PowerWater Sterivex Kit Handbook]]
 . Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.

@@ Line 47: / Line 47: @@
 .
-.
+. [[file:///C:/Users/saras/Downloads/HB-2266-002_HB_DNY_PowerWater_Sterivex_0519_WW%20(2).pdf|DNEASY PowerWater Sterivex Kit Handbook]]
 . Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.

← Older revision		Revision as of 05:15, 20 September 2021
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−	7. Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. ~~<nowiki>[https://journals.asm.org/doi/epub/10.1128/AEM.01043-13</nowiki>~~ Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform]. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.	+	7. Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.

← Older revision		Revision as of 05:11, 20 September 2021
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	6.		6.

−	7. Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.	+	7. Kozich, J. J.; Estcott, S. L.; Baxter, N. T.; Highlander, S. K.; Schloss, P. D. <nowiki>[https://journals.asm.org/doi/epub/10.1128/AEM.01043-13</nowiki> Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform]. Applied and Environmental Microbiology. 2013. 79 (17). 5112-5120.

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 ===DNA Extraction===
-DNA extraction from the 0.22 um sterivex filter unit followed the protocol outlined in the DNeasy Powerwater Sterivex Kit with no noteworthy deviations from this protocol (). As expected, 100 ul of product was obtained. 2 ul were tested on the nanodrop to assess the quantity and quality of the DNA obtained. The DNA extracted from the freshwater sterivex filter unit absorbed at a concentration of 3.6 ng/ul at 260 nm, which was a lower concentration of DNA than expected. Its A260/A280 ratio was 1.80, which indicates that the protein and inhibitor removal steps were successful. The recorded A260/A230 ration was very low at 0.04, which may indicate the presence of excess salt left in solution, for most of the salts used in this DNA extraction protocol absorb at 230 nm. This low ratio suggests that improvements to the overall quantity of DNA obtained may be higher than 3.6 ng/ul through purification techniques aimed to get the excess salt out.
+DNA extraction from the 0.22 um sterivex filter unit followed the protocol outlined in the DNeasy Powerwater Sterivex Kit with no noteworthy deviations from this protocol (6). As expected, 100 ul of product was obtained. 2 ul were tested on the nanodrop to assess the quantity and quality of the DNA obtained. The DNA extracted from the freshwater sterivex filter unit absorbed at a concentration of 3.6 ng/ul at 260 nm, which was a lower concentration of DNA than expected. Its A260/A280 ratio was 1.80, which indicates that the protein and inhibitor removal steps were successful. The recorded A260/A230 ration was very low at 0.04, which may indicate the presence of excess salt left in solution, for most of the salts used in this DNA extraction protocol absorb at 230 nm. This low ratio suggests that improvements to the overall quantity of DNA obtained may be higher than 3.6 ng/ul through purification techniques aimed to get the excess salt out.
 [[:File:PowerWater_Sterivex.pdf]]
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 Reverse primer design: 5'-CAAGCAGGAAGACGGCATACGAGAT-AGTCAGTCAG-CC-GGACTACHVGGGTWTCTAAT-3' | 5'-adaptor-pad-linker-V4 region primer-3'
-The pads serve the purpose of preventing hairpin formation and manipulating the melting point of the primers, for the adaptors need to anneal to the flow cell, a glass slide with lanes of two different types of oligo (complementary to the adaptors), at 65°C in order for sequencing to ensue (). The linkers are noncomplementary to the 16S gene so that alignment for bioinformatics processing can be carried out efficiently (). The adaptors, pads, and linkers are needed on both the forward and reverse primers because bridge amplification is utilized in Illumina sequencing platforms. During this process, one side of the adaptor sequences hybridizes to the flow cell, a complement of this original fragment is produced by a polymerase, the double strand of DNA is denatured and washed away, and then the single strand folds over and hybridizes to the  second type of oligo on the flow cell (). Polymerases generate the complementary strand, and this double stranded bridge is denatured, which results in two single stranded copies tethered to the flow cell (). This process is repeated many times. After bridge amplification, the reverse strands are cleaved and cut off, which is why the barcode is only important for the forward primer.
+The pads serve the purpose of preventing hairpin formation and manipulating the melting point of the primers, for the adaptors need to anneal to the flow cell, a glass slide with lanes of two different types of oligo (complementary to the adaptors), at 65°C in order for sequencing to ensue (7). The linkers are noncomplementary to the 16S gene so that alignment for bioinformatics processing can be carried out efficiently (7). The adaptors, pads, and linkers are needed on both the forward and reverse primers because bridge amplification is utilized in Illumina sequencing platforms. During this process, one side of the adaptor sequences hybridizes to the flow cell, a complement of this original fragment is produced by a polymerase, the double strand of DNA is denatured and washed away, and then the single strand folds over and hybridizes to the  second type of oligo on the flow cell (8). Polymerases generate the complementary strand, and this double stranded bridge is denatured, which results in two single stranded copies tethered to the flow cell (8). This process is repeated many times. After bridge amplification, the reverse strands are cleaved and cut off, which is why the barcode is only important for the forward primer.
 ===PCR Amplification===
@@ Line 37: / Line 37: @@
 ==References==
 . Savage, D. C. [[Media:32_300.pdf|Microbial ecology of the gastrointestinal tract.]] Ann Rev Microbiol. '''1977'''. ''31''. 107-133.
 . Smith, K. F.; Schmidt, V.; Rosen, G. E.; Amaral-Zettler, L. [[Media:pone.0039971.pdf|Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water.]] ''Public Library of Science One.'' '''2012'''. ''7''.
 ==Page History==
 This page was created by Sara L Simonson in the spring of 2021