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ISSR Genomics

Lab Entry 13 – Genomics

For this lab, analysis was done of the lupin plant DNA collected and utilized in previous posts. To run analysis, first a tutorial was completed in Geneious on reference-based assembly and de novo assembly. This was performed with sample sequences developled by Geneious and downloaded from the online website and installed into the Geneious program. A questionnaire was completed along with taking screen shots of the final results from the tutorials of both reference-based and de novo assemblies.

Reference-based Assembly

  1. SRR10, SRR11, SRR18, SSR23, SSR25
  2. Paired-end because it makes direct alignments of the reads and trims the reads while using a reference.
  3. It took very little time.
  4. Ave. 98.1, min. 1, max. 139, the ends have the lowest coverage because they are the variable at the ends with various base pair lengths.
  5. 4035 G –> K, 3961 A –> W, 3972 T –>D, 3803 T–>W, this tells us that there are polymorphisms at this location.
  6. 3798-3807, they show little significance and hence, exclude them
  7.  207 G to C (transversion – protein substitution), 222 G to A (transition), 230 C to T (transition), 311 T to A  (transversion – protein substitution)

De Novo

  1. 25172 reads total, 4 contigs, 17994 in largest, 511 minimum length, 133444 mean, 192891 NC50 score
  2. 27.7 secons, contig 1 = highest coverage, contig 3 = lowest coverage
  3. 92077-92267
  4. 30.75 seconds
  5. 340, 18033, 58852

The final aspect of this lab was using binary code to create a Nexus file of the data results from running gel electrophoresis of the lupin DNA samples. A file was created based on a template provided by Prof. Paul. My data was substituted into the file in which the plant sample names were put into the file with respect to the presence of bands based on the gel results. For each sample that displayed a band in a row, the sample would be marked as a ‘1’ for that row. If there was no band present, a ‘0’ would be assigned, or if the band was questionable if it was being displayed of not, a ‘?’ was assigned for that particular band. This was done for all of the samples between Omar and 17898 gel electrophoresis runs excluding some of the samples that displayed insufficient data.

Here is the Nexus file in the form of a .txt of the set up to be used in  the next lab.

ISSR_data_format_mydata-1jpblln

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Analyzing Lupinus ITS Sequences

Lab Entry 12  – Analysis of ITS Sequences

The first part of this lab was to prepare for lab 13. Using the samples from last lab with Omar and 17898 primers, three randomly selected samples were chosen to be used for gel electrophoresis. PRA03, PRA04, and PRA05 were the chosen samples to be used. 1 µL loading dye was added to each of the samples and then run on a large gel containing 40 wells, one gel for Omar primers and the other for 17898 primers. The gels were run at a low voltage (60 volts) for approximately 1.5 hours. Once the gels were completed, they were imaged and then saved to be used for the next lab.

Analysis

The majority of this lab was working on an alignment of lupine ITS sequences that were conducted from the last lab using Geneious. Forward and reverse reads of each of the plant samples were used, excluding any plant samples that did not have reads with significant information on them.

The first step was to create a consensus sequence of the forward and reverse sequences for each of the plant samples. By selecting the forward and revers sequences of one plant sample, the sequences were aligned using a De Novo Assembly. The defaults were kept the same and the alignment was created. Within the assembly, the ends were trimmed to create a coherent alignment by deleting ‘bad’ regions of the sequences. Any incorrect base calls were also edited by comparing one sequence to the other sequence and making a decision about the base pair that should be there. With that finalized, the consensus sequence was chosen and then extracted into a new file that was renamed. This provides the consensus sequence for that one plant sample. This process was conducted for all of the plant sample forward and reverse sequences until all 41 samples had their own consensus sequences.

The next step was to create an alignment of all of the consensus sequences. All of the consensus sequences were selected and then aligned using the Muscle tool. Once the alignment was made, similarly to the above sequences, it was edited and the ends were trimmed to make a more coherent set of sequences.

The last step was building a phylogenetic tree using Mr. Bayes under certain conditions with Lcham as an outgroup for the tree. Once the tree was completed, analysis took place regarding the process of ITS and how the plant samples are related to each other, specifically the relatedness of plant samples with yellow flowers versus purple flowers.

Results:

There was one clade with support > 0.85 and the samples withing that clade are geographically close to each other. However, there is another PSF sample that should be included within the clade as well. Between the yellow and purple flowing lupines, there was no pattern displayed that shows their relatedness since there is not a clade of only purple of yellow flowering lupines. Rather, there are interspersed within any of the clades with the exception of the clade with >0.85 support.

It appears that many of the lupines are genetically very similar to each other and hence, such a large selection of the samples deriving from a single node. I can’t say for sure if this is due to ITS resolution or potentially a mistake with the samples that was made. If it is due to resolution of ITS, the argument can be made that the samples were not distinguished to a high enough level since there was so much crossing between the yellow and purple flowing lupines. Again, it could also be due to mistakes made in the lab that caused this issue. Another interpretation is that color may not be affected by the particular sequence selection which in turn, would result in a tree that mixes the yellow and purple lupines.

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Plant DNA PCR – ISSRs

Lab Entry 11 – ISSRs

Two interspersed-simple-sequence-repeat (ISSR) primers were used in PCR reactions.

First, dilutions were made of the template DNA of the plant samples to aid with the success rate when performing PCR. In 1.5 mL tubes, 10 µL of each DNA samples was added to 90 µL of ddH2O to create a 1 : 10 dilution. Two 0.2 mL 8-strip tubes were used where each sample would be placed into one of the tubes on each strip because two PCR tests were run. With all of the tubes labeled with the correct plant ID identification, 1 µL of template DNA was added to each corresponding tube so that  both tubes had the exact same elements within them.

Two master mixes were needed in order to run two PCR tests. To make the master mix, we calculated each ingredient to make enough for 20 reactions for my table group so we would have some excess master mix. For both mixes, 250 µL ddH2O, 60 µL 10x bufer + Mg, 20 µL BSA, 40 µL dNTPs, 5 µL Taq, and 5 µL of primer was used to creates the mixes. The primers being used were Omar and 17898 as they displayed the best results after running the samples through gel electrophoresis. Therefore, 5 µL of Omar was added to one master mix and 5 µL of 17898 primer was added to the other master mix.  Both mixes were vortexed to ensure the components were mixed well within the tubes.

To each of the tubes PCR tubes containing 1 µL template DNA, 19 µL of master mix was added. Master mix with Omar was placed into a single strip while 17898 was added to the other strip. The tubes were labeled with an “O” or “#” to distinguish between Omar and 17898. The tubes were then placed into separate areas to be run through PCR.

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Plant PCR II

Lab Entry 10 – Plant PCR II

Plant DNA PCR…Round 2

Due to some unfortunate results from the previous attempt at plant DNA PCR, the experiment was repeated. The primers being used were from the chloroplast gene psbA, were the same as the last time. The procedure that was performed was the exact same as completed in the first Plant PCR post (Lab Entry 9). For this experiment, extra attention was given to the amounts of solutions being transferred using pipettes as that was likely the cause of the problems from the fist run through of the experiment. The only other difference were the samples used. The samples I tested were PRL01, PES05, PRL05, PSF01, and PRA01.

Plant DNA PCR – ISSRs

The same samples listed above were used for this portion of the lab. The class tested four interspersed-simple-sequence-repeat (ISSR) primers in PCR reaction and my table group tested the Omar primer.

The same PCR tube strips were used as with the psbA experiment in which 1 µl of each of the samples were placed into their labelled tubes. The tubes were set aside while a master mix solution was put together.

The master mix was used among the table and was set up for a total of 20 reactions. To make the master  mix, 250 µl ddH2O, 60 µl 10x buffer +Mg, 20 µl BSA, 40 µl dNTPs, 5 µl Omar primer, and 5µl Taq was all added to together and mixed thoroughly. Once the master mix was complete, 19 µl was added to each of the DNA samples. The lids on the tubes were closed tightly and then place into the PCR machine. Analysis of the DNA will occur in the following lab entry post.

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Plant PCR I

Lab Entry 9 – Plant PCR I

Gel Electrophoresis

The first portion of the lab was taking the extracted plant DNA from the last lab entry and running it on a gel. 1.0 μl of loading dye was placed onto parafilm five times to make 5 dots on the parafilm. To this, I added 4.0 μl of each plant DNA template sample to one of the dots. These dots were then taken up individually and added to the gel with other people’s samples as well. Two ladders were placed at the beginning of each of the rows and my samples were placed in wells 2-6 on the lower layer of the gel. The gel was run using 130 volts for 25 minutes.

PCR of the plant samples was then completed to continue the lab and amplify the sample DNA. For the PCR setup, primers for two markers were used: internal-transcribed spacer (ITS) and a chloroplast gene (psbA). I used two 0.2 ml 8-tube strips (PCR tube strips) for this portion of the lab. After labeling both tube strips with the plant ID’s discuss in the previous post, 1 µl of each of the plants samples was placed into the respective tube for both tube strips using filtered pipette tips. Once all of the tubes contained template DNA, the lids were placed on top and set aside.

Two master mix solutions were created for the plant samples in order to use primers for the two markers introduced earlier, hence the two tube strips being used. The amount of reagents was first created that would provide enough master mix for my table group. This resulted in each master mix possessing: 300 µl ddH2O, 40 µl 10x buffer + Mg, 20 µl BSA, 4.0 µl dNTPs, 4.0 µl of forward and reverse primer each (for either ITS or psbA), and 0.8 µl Taq. This would allow 19.0 µl of master mix to be used per sample for a total of 20 µl.

Master mix with ITS primers was added to each tube for one of the tube strips and master mix with psbA primers was added to the second tube strip. Once all of the tubes had either master mix for ITS or psbA, the lids were placed on top of the tube strip securely and were then ready for the PCR machine. The tube strips were separated for either ITS or psbA for the class for two PCR runs to occur.

Pictured above is the gel electrophoresis results. As mentioned, my samples were on the lower level of the gel in wells 2-6. All of the samples ran well through electrophoresis which will in turn, (hopefully) produce good results for the PCR portion of the lab.

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DNA Plant Extraction

Lab Entry 8 – DNA Plant Extraction via Modified Alexander et al. Tube Protocol

This lab is the continuation of the past field trips that were taken in which various plant samples were collected around the Bay Area. The samples assigned to me originated from Abbotts Lagoon in Point Reyes. For all of the labeling, the code ‘PRA’ was used followed by the 01-05 for each of the five samples used during the lab.

1.5 mL centrifuge tubes were labeled PRA01, PRA02, etc., for the five plant samples collected from my assigned location. A small amount of leaf tissue was added to each of the tubes accordingly along with three 3.2-mm stainless steel beads and were placed in a tube rack along with some of the other students’ designated plant samples as well. The rack was then loaded into a modified reciprocating saw rack mount and was reciprocated on speed 3 for approximately 40 seconds.

The tubes were then spun down in the centrifuge for 15-20 seconds to pull the plant particles to the bottom of the tube. Approximately 434 μl of preheated grind buffer was added to each of the tubes and incubated at 65 °C for 10 minutes in a water bath, mixing the tubes every three minutes or so.

Once finished in the bath, 130 μl 3M pH 4.7 potassium acetate was added to each of the tubes and inverted to mix before being placed on ice to incubate for 5 minutes.

After incubating, tubes were centrifuged at maximum force for 20 minutes. New 1.5 mL tubes were labeled with the same sample ID as before. Any supernatant at the top of the centrifuged tubes was transferred out using pipettes, carefully avoiding transferring any of the precipitate. To these new tubes, 1.5 volumes of binding buffer was added for each of the tubes based on the volume of supernatant that was acquired. I added anywhere from 400 to 500 μl of buffer to the tubes with one tube needing less due to a lower volume of supernatant being acquired.

 

650 μl of the solution (or all of the solution if the volume was less than 650 μl) from the tubes was transferred to Epoch spin column tubes and were centrifuged for 10 minutes at 15,000 rpm in order to collect the DNA in the silica membrane filter as the buffer drained through the filter into the collection portion of the tube below. The buffer left in the tube was discarded and the filtered portion was added back to the tube. The tubes were then centrifuged again for another 10 minutes, and again, any liquid at the bottom of the tube was discarded.

DNA bound to the silica membrane filter was washed by adding 500 μl of 70% EtOH to the column and centrifuged for 8 minutes which would pass to the collection tube. The flow-through was discarded. Tubes were centrifuged again for approximately 5 minutes to remove any residual ethanol. The collection tubes were discarded and the columns were placed in sterile 1.5-mL microcentrifuge tubes that were also labeled with the plant codes on them.

To the new tubes, 100 μl preheated (65 °C) pure sterile water was added and were let to stand for about 5 minutes. After standing, the tubers were placed in the centrifuge and ran for 2 minutes at 15,000 rpm to elute the DNA. The tubes for the class were then all collected to be stored for the next lab session.

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Geneious II

Lab Entry 7 – Geneious Part II

Using the sequences acquired from the last lab entry (30 sequences in total), phylogenetic trees were created using various programs within Geneious. The first step to creating the trees was to align all of the sequences and then ‘clean’ them so that all of the sequences begin and end at the same point.

The next task was to create a model of molecular evolution using a program called jModelTest. I was unable to utilize this program because I use Windows/PC rather than MAC. Because of this, I observed Ryan Aquino go through the steps which began with placing the aligned sequence in the jModelTest sequence. The program runs going through 88 models and calculates likelihoods for each of those models. Once the program is done running through the various models, the best model is chosen using two models. The first is the Akaike Information Criterion  and Bayesian Information Criterion which I was also unable to perform without the jModelTest.

I could run the MrBayes program within Geneious which was the next task to create a phylogenetic tree. Using the same aligned sequence that would’ve been used for the jModelTest, the sequence was run through the MrBayes program with specific parameters provided. The result was a tree, as well as two other graphs. These graphs provided indication that the program was not run long enough and I was instructed to run the program with different parameters that would allow the program to run for a longer duration. This resulted in a very different tree that was much more accurate based on comparing the species used in the alignment. The longer MrBayes run was performed after the lab session when more time was available.

The last step of the lab was to run programs for the maximum likelihood. There were two ways to do this: RAxML and PHYML. My computer would not run PHYML but I could run the RAxML program. The result was a tree different than the one provided by MrBayes. This tree was then run through a program to make a consensus tree. The RAxML tree was less accurate in comparison to the MrBayes tree. The timeliness of each of the programs displays the quality of the tree being produced. RAxML was very quick and showed some accuracy with relationships of species but the MrBayes program took much longer and was clearly more accurate when comparing species to one another and how they relate to each other.

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Geneious

Lab Entry 6 – Geneious Gene Sequencing and Analysis

With the completion of DNA replication and running multiple PCRs, results of gene sequences were received in which analysis could take place. A program, Geneious, allows for this analysis by comparing the forward and reverse sequences of data found for each of the samples. All three of my samples produced sequences with high HQ% values. With all six of the forward and reverse sequences placed into a file on Geneious, a comparison could be made for each of the three samples.

First, the two matching sequences were selected and then ran through De Novo Assembly to piece the sequences together to match each other as accurately as possible. The program runs the forward and reverse sequences in the same direction for a comparison to occur. Any base pairs that were unable to be sequenced properly were deleted while some base changes were altered in order to match the other sequence based on the level of accuracy produced by the program. Once the sequences were trimmed and corrected to match each other, the sequence was compared to other sequences withing the Geneious  in order to determine the origin of the sample. The program lists a multitude of potential matching sequences, providing a percentage of how closely they relate to each other. The sequence with the highest sample can be assumed to have the same DNA as the sample tested resulting in the genetic identification of the species of sample.

Exercises:

1). All of the samples were correct when comparing sequence identification to what the samples were marketed as in the restaurant. Chinook salmon, yellowfin tuna, and yellowtail were all correctly identified from the sequencing performed.

2). The salmon sample had no polymorphic sites when comparing my sequences to the sequences of the best matching species. The tuna sample had quite a few polymorphic sites where there were different base pairs at the same location. The yellowtail sample had two polymorphic sites near the end of the sequences but matched perfectly other than those two differences.

3). For the third part, 27 sequences were collected to go along with the three sequences from my samples for a total of 30 sequences to compare with. The sequences consisted of ray-finned fish species (Actinopterygii) and a few shark and ray species (Chondrichthyes).

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Field Trip II

Lab Entry 5 – Field Trip II

 

For the second filed trip the class took, we ventured to the Limantour Trailhead area in Point Reyes National Seashore. This location was approximately 1.5 hours north of San Francisco via driving. Lupinus arboreus was found on the trail to the beach with a distinct difference that the plants found in the Half Moon Bay area from the last field trip taken. Lupinus arboreus at this northern location consisted of flowers with a purple color rather than yellow as seen in Half Moon Bay. This was the only visible difference between the plants in the north from the south. In addition to viewing our Lupinus arboreus, wildlife was also seen including seals and humpback whales. Samples of Lupinus arboreus were collected prior to the field trip by the Professor Paul .

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Gel Electrophoresis

Lab Entry 4 – Gel Electrophoresis

A template organizing where the samples were placed was provided in which each of the samples for the table was designated a specific lane to be place in. The gels being used have two rows with 8 lanes in each. The top row of gel was occupied by other table-mates and a ladder while the bottom row consisted of my three samples and another ladder. The gel was loaded in accordance to this template.

gDNA Electrophoresis

With a 1% agarose gel with GelRed already cast, the gel tray was placed into the gel box with the top of the gel at the negative (black) end of the electrical connector. 1x TAE buffer was poured into the gel box so that there was enough to cover the gel by a couple millimeters.

On a piece of parafilm, 2.0 μl Loading Dye was dropped for the number of samples being used. In our case, we placed 12 drops on the parafilm to be used for each of the samples for the table. Approximately 3.0 μl of gDNA was added to the Loading Dye for each of the samples. This was done until all dots were completed with Loading Dye and gDNA.

The pipette was readjusted to 5.0 μl and then each drop was pipetted into the designated well on the gel for all of the samples of gDNA. Once all of the wells were filled with their designated samples, the lid was placed onto the gel box with positive and negative charges aligned and then turned on. The volts were set to 145 and the gel ran for approximately 16 minutes before the power was turned off.

The gel was then imaged using the Gel Doc EZ Imager.

Making 1% Agarose Gel

For the gel used for the PCR electrophoresis, the gel previously used for the gDNA run was melted down in a microwave to be reused for the PCR run. The gel was placed into a beaker and placed in a microwave for approximately 25 seconds, allowing the solution to bubble for about 10 seconds. To the liquid, another 1 μl of GelRed was added to the beaker in case of any loss. The gel was then poured into the casting rig set up to make the gel with the two rows of wells using combs. The gel was left to harden for about 10 minutes to be used for the PCR electrophoresis run.

PCR Electrophoresis

For the PCR electrophoresis run, the same procedure was performed as for the gDNA electrophoresis run. All of the measurements were the same as well as the entirety of the process of dying and placement of samples on the gel. The only difference was the addition of a negative control that was made in the previous post which was then placed into one of the wells on the gel. Voltage and time for the electrophoresis run were the same as for the gDNA run.

Exo-Sap Clean-up

For the clean up process, we used RA02, RA03, JPCC, JPCD, JW01, JW02, & JW03. These lanes had the best results for banding based on the computer imaging of the PCR gel plate. A master mix of ExoSap solution was created for all of the table-mates. Enough solution was created so that 9 clean-ups could be performed from the best samples and extra solution in case it was needed. The master mix consisted of 95.31 μl water, 11 μl 10x buffer (Sap 10x), 3.96 μl SAP, and 1.98 μl Exo. 7.5 μl of PCR product was placed into 0.2 μl PCR tubes for each of the samples in addition to 12 μl of the ExoSap master mix. Once all tubes contained the necessary amounts of products, they were placed into a thermocycler to undergo the EXOSAP program which takes approximately 45 minutes.