Course: DNA Fingerprinting



DNA Barcode Data Analysis of Your PlantsSample (gather some info about the plant you isolated DNA from)If you think you know your plant, find some info on it, Wikipedia is a good place to startEnter the data into the table below Sample NoCommon nameOrderFamilyGenusSpeciesSequence quality (examine the quality of your sequences)Open DNA Subway Log into DNA Subway using your Username and PasswordClick on the blue squareUnder Barcoding click on rbcLClick on Import Trace Files from DNALCSearch DNALC data for Nadja AndersonClick on the Tracking #Click on Select AllOn the bottom of the window click on Add Selected FilesWait until files are loadedEnter a Project titleOptional: enter a Description for your projectClick on ContinueClick on Sequence ViewerMaximize the Sequence Viewer window by clicking the square in the upper right-hand cornerThe first column indicates sample IDs, allows you to view the sequence for quality (is your sequence quality good?).Use the horizontal bar at the bottom of the window to slide the sequences back and forth; notice that the sequences are quite long.Find your sample.Examine your sequence by sliding it horizontally – is your sequence framed by “N”s? What do you think N’s stand for?How long is your sequence? Does the electropherogram resemble that of a good sequence?Close the Sequence Viewer windowClick on Sequence TrimmerClick on Sequence Trimmer againMaximize the screen and locate your sample again – is it still framed by “N”s?Move your cursor over the icon following your sample ID – did it receive a “Low Quality Score Alert?Click on the sequence icon (2nd column) and examine the trace file for your sequenceCompare the trace for your sequence to others Click again on the icon for your sequence and examine the trace in detailHow high is the amplitude for your sequence?At how many places do traces overlap each other?How many “N”s are in your sequence?Is your sequence of consistent quality across its entire length?Record the Sequence quality of your sequence in the table below.How does the quality your sequence compare to others?Sequence QualityID #Warning Yes or NoAmplitude Low, High or MediumLengthOverall QualityIf you sequenced in both directions, you can pair your sequences. If you did not skip this step andgo to Reference data below. Pairing the forward sequence to the reverse sequenceClick on Pair Builder and select your forward and reverse sequence (you will need to designate the reverse sequence by changing the F to an R by clicking on it). When asked if you would like to pair these two sequences select Yes then Save.Notice Consensus Editor is not ready to run whereas before it was blocked. Click on Consensus Editor.Click on Consensus Editor again once you can view the results.You will see the alignment of your DNA sequence in the forward direction and the reverse compliment. Should these two align without mismatches, then the confidence for your sequence should be twice as high.You can either unpair your sequences for the following alignments and BLAST searches with your sequences, or keep them paired. Keep in mind that if they are paired you will be working with the forward sequence combination of the two sequences, however, if your sequences aligned well the this should be the same as your forward sequence. Reference data (add reference sequences to compare your plant sequence against)Click on Reference DataClick on Common plants Click on Add ref dataClose the Reference DataYou may choose to do this again with Eudicots and MonocotsDetermine sequence similarities (align and compare your sequences against references)Click on Select DataCheck your sampleCheck an entire reference data set or a subset of plants from this listClick on Save SelectionClick on MUSCLEAfter the button changed from “R” to “V” click on Muscle again to view the alignmentClick on ATCG to zoom into the multiple alignmentPan horizontally and try to find what reference sequence your sample sequence resembles best; write it down in the table belowClick on Trim Alignment – what happens? (Be patient, trimming may take time)Close the Alignment ViewerTo compare your sequence with to the reference data, click on PHYLIP MLAfter the button changed from “R” to “V” click on PHYLIP ML againWhat does the tree depict?Determine what reference is most closely related to your sample and write it down in the table belowDownload an image of the tree to your computerClose the PHYLIP ML windowResults from Reference comparisonBest alignment match2nd best match3rd best matchBest match in tree2nd best match3rd best matchRepeat the alignment and building of a tree with all of the sequences, you do not need to tabulate the information for each of the sequencesDoes your sample aligned more closely with one of the other sequences? BLAST search (identify additional reference sequences through a sequence search)Click on BLASTNFor your sample click on BLASTThe BLAST results will be returned in a new windowExamine the different matches and record the Details, Aln length, Bit score, e-value and Mismatches for different organismsClick on the species name for the first match, then for othersWhich do you think matches your species most closelyWhen you click on the species name a new box pops up with Wikipedia information, click on more to open this in a new windowWhat is the species, if there is a picture, does it look anything like your plant?In the table at the very beginning of this worksheet fill in the appropriate classification for this plantCheck the boxes of three different BLAST hitsClick on Add BLAST hits to projectRe-Determine sequence similarities (align and compare)Click on Select Data (remember to scroll down to see the BLAST hits)Check the BLAST hits to include them in the analysisClick on Save SelectionClick on MUSCLEAfter the button changed from “R” to “V” click on Muscle againClick on Trim Alignment (be patient, trimming may take a little while), what aligns most closely to your Close the Alignment ViewerBuild a phylogenetic tree with PHYLIP MLAfter the button changed from “R” to “V” click on PHYLIP ML againWhat does the tree depict?Determine what reference is most closely related to your sample and record the information belowDownload an image of the tree to your computerClose the PHYLIP ML windowResults from BLAST searchesBest alignment match2nd best match3rd best matchBest match in tree2nd best match3rd best matchSequence comparison of class plants (align and compare your sequences against the class sequence)Click on Select DataCheck user data (you may want to eliminate some of the more questionable data, and you may want to just look at your groups which will be either A or B)Click on Save SelectionClick on Sequence Trimmer to trim the sequencesClick on MUSCLEAfter the button changed from “R” to “V” click on Muscle again to view the alignmentClick on ATCG to zoom into the multiple alignmentPan horizontally and try to see if any of the classes’ sequences share similarityClick on Trim Alignment – what happens? (Be patient, trimming may take time)Close the Alignment ViewerTo compare these sequences to each other, click on PHYLIP MLAfter the button changed from “R” to “V” click on PHYLIP ML againWhat does the tree depict?Determine which of the classes’ data is most closely related to your sample. Were any of the samples the same species? What evidence allowed you to come to this conclusion.Download an image of the tree to your computerBLAST search and compare sequence similarities for any of the sequencesYou can go through the above sequences and determine their most closely related species using BLAST as you did for your sample aboveAdd BLAST hits to projectSelect that dataMUSCLEPHYLIP MLDownload tree to see how the other sequences compare to the your data and to the BLAST hitsYou can add the reference Data and see if any of the samples are closely related to the reference groupNow that you have completed the Plant DNA Barcoding activity, how could you see this being used? Provide one possible way that barcoding for plants could be useful for our society or for a commercial endeavor. Do you think barcoding could be useful for other species besides plants? Provide a useful exploit for barcoding of other species. ................
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