TITLE: Fungal isolate identification using ITS-LSU regions
AUTHORS: Cassie Ettinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was written as a companion to the </span><a href="https://peerj.com/articles/960/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">'Swabs to Genomes'</span></a><span> workflow to enable undergraduate interns to take Sanger sequences for the ITS-LSU region from unidentified fungal isolates associated with seagrasses and identify preliminary taxonomy via databases searches and then build phylogenies to further assess putative identities.</span></div></div>

[STEPS]
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
If on undergraduate computer in the Eisen lab, you can type in ‘seqtrace’ into ‘terminal’ to start. Otherwise you may need to type 'python seqtrace.py'
Follow directions here on how to install and use Seqtrace: https://peerj.com/articles/960/#sanger-sequence-processing.
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
Create a new project (File > New Project).
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
Choose the directory your Sanger sequences are in.
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
To add files, go to “Traces” and click on “Add trace files”, then select the reads (.abi files) you want to work with.
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
If your files have the primer names in them, you can enter their names under ‘search strings…”.
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
Add your forward and reverse primer names (sequences not necessary).
Possible forward primers: ITS5, ITS1F.Possible reverse primers: LR3, ITS4.
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
Now click on the ‘Sequencing Processing’ tab.Under “Sequence trimming”, unclick the 'Automatically trim sequence ends' button.Under “Consensus” decrease the min. confidence score to 15.
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
Group your forward and reverse reads by highlighting both of them and clicking “Group selected forward/reverse files” (under “Traces”).
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
Under “Sequences” go to “Generate Finished Sequences” and click on “for all trace files”.
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
To view your consensus sequence, click on the read pair group and then click on the magnifying glass at the top of the page.
The “Trace View” shows the quality scores, the chromatogram (trace) display, and the raw base calls from both the forward and reverse reads, as well as the consensus sequence. The consensus sequence is the middle list of nucleotides.
?. [Process Sanger sequences following the ‘Swabs to Genome’ workflow using Seqtrace]
To export the consensus from the trace view, go to “Sequence”, hover on “Export Sequences”, and select “Export Sequences from Selected Trace Files”.
This will create a file containing the consensus sequence.
?. [Identify fungal sequences using existing databases]
Go to NCBI's BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
?. [Identify fungal sequences using existing databases]
Click nucleotide blast.
?. [Identify fungal sequences using existing databases]
Upload the fasta file containing your consensus sequences or copy / paste them in the box.
?. [Identify fungal sequences using existing databases]
For each sequence write down the top hit & its % identity (this is how much the top hit matches your sequence). Note that the top match is database dependent, and for our purposes here is just a hypothesis. We will check our sequences against several databases and build a tree to determine how confident we are in this hypothesis.
?. [Identify fungal sequences using existing databases]
Go to Ribosomal Database Project (RDP) website (https://rdp.cme.msu.edu/).
?. [Identify fungal sequences using existing databases]
Make your way to the “Classifier” (https://rdp.cme.msu.edu/classifier/classifier.jsp).
?. [Identify fungal sequences using existing databases]
Upload/paste your sequences.
For “choose a gene”, we are selecting what database to use for ITS1-ITS4 this is UNITE; for ITS5-LR3 you will want to do this process three times, once using UNITE and once using “Fungal LSU training set”, and one using “WARCUP”.
?. [Identify fungal sequences using existing databases]
Click on “Show assignment detail for Root”.
Stop here and decide on next steps! There are now two options to build fungal phylogenies, the first is to try to build a tree using SILVA (described below), but this is not very effective for fungi that SILVA does not have lots of data for (e.g. works well for Penicillium, but not for more novel or less studied taxa). The second way is to build a phylogeny manually - which involves (1) downloading fungal sequences to include in the tree from BLAST and the literature (2) generating an alignment (3) trimming the alignment and checking it manually (4) picking an evolutionary model for tree building and (5) building a phylogeny. Steps (2), (4), and (5) can be performed on CIPRES (https://www.phylo.org/portal2/home.action).
?. [Build a tree for the LSU region (if available)]
Go to SILVA ACT (https://www.arb-silva.de/aligner/).
?. [Build a tree for the LSU region (if available)]
You will need at least 4 sequences to build a tree - so wait until you have 4 sequences or add in sequences you already have to get to 4.
?. [Build a tree for the LSU region (if available)]
Pick a named bacteria, click its name to add it to your cart and then click “Download” to get the sequence as        a fasta file.
We want to add in a bacteria as an outgroup so you actually only need 3 fungal sequences to build your tree. We can download an outgroup from https://www.arb-silva.de/browser/.
?. [Build a tree for the LSU region (if available)]
Under “Basic alignment parameters”.
?. [Build a tree for the LSU region (if available)]
Paste at the end of your fasta file or add to box if copy/pasting.
?. [Build a tree for the LSU region (if available)]
Alternatively use the bacterial sequence below:>OUTGROUP Bacteria;Chloroflexi;Chloroflexia;Chloroflexales;Chloroflexaceae;Roseiflexus;Roseiflexus castenholzii DSM 13941AAGACAGAAGGAGCACACGGAGGAUGCCUCGCCGCUGCGCGCCGAUGAAGGACGCGGCAACGCUGCGAAAAGCGUGGGGGAGCCGCGCGCAGGCUGUGAUCCCACGAUGUCCGAAUGGGGCAACCCGUCCGUGAUACGGACACCGCGCAAUGCGGAGGGCACCGGGGGAACUGAAACAUCUUAGUACCCCGAGGAACAGACAGCAUCCCCGGAGUAGUGGCGAGCGAAACGGGGUGAGCCCAAACCACGACCGUGGCAAGGCGGCAGCCGUUGCGGUCGUGGGGUUGAAGGGAGCGCUGCGGGGGGACUGCCGACCCCCCCGCGCGCGUCGCACCCGGGCGACGAAGCCGACUGGAAAGCGGCGCCACAGAGCGUGACAGCCGCGUAGUCGGGCGGGUGCGGCGGCGCGAGGCGCUUGCCUGAGUAGCGCCGGACACGAGCAACCCGGCGUGAAACUGGGGCGACCACGCUCCAAGGCUAAAUACGCGCAGCGAGCGAUAGCGAACGAGUACCGUGAGGGAACGGUGAAAAGCACCCCGGCGAGGGGAGUGAAAGAGAACCUGAAACCGUGUGCUUCCAUGCAGUCGGAGCCCCCGUGAGGGGGUGACGGCGUGCCUUUUGGAGUAUGAUCCGGCGAGUUACCCUCGGUGGCCAGGUUAAGGCAGUGACAGCCGGAGCCGGAGCGAAAGCGAGUCUGAAGAGGGCGCGCGGUCGCCGGGGGUAGACCCGAAACCGCUUGAGCUACCCAUGGGCAGGGUGAAGCGCGCGUAACAGCGCGUGGAGGCCCGCACCGGUGUGGAUUGCAAACCGCUCGGAUGACCUGUGGGUAGGGGUGAAAUGCCAAACGAAAGCGGAGAUAGCUGGUUCUCCCCGAAAUGCAUUGAGGUGCAGCCUGCGGAAUGCCGCGCGCGGAGGUAGAGCGCUGGUGUGGUGCGGGGGCUUCACCGCCUACCAAACGACGCCAAACUGCGAAUGCCGCGCGGGGAAGCGCAGAGUGAGACGUGCGGCGCAAACGUUGUACGUCGAGAGGGAAACAACCCAGACCUGCAGCUAAGGUCCCGAAAUCGACCCUGAGUGGGAAAGGAUGUGUCGUUGCGGAGACAACCAGGAGGUUGGCUUAGAAGCAGCCACCCUUGAAAGAGUGCGUAACAGCUCACUGGUCGAGUGACGAUGCGCCGACAAUCCAGCGGGGCAAAGGGUCGUACCGAAGCUCAGGACCGGAAGGGUGGUAGGGGAGCGUCGUGGACGCGGCGAAGCGGCGGCGGAAGCCGGUCGUGGAGCGUCGACGAGUGCGAAUGCCGGAACGAGUAACAGCGAAGCGGGGUGCGAACCCCCGCCACCGGAAGCCUCAGGGUUCCGCCGCAAGGGUGAUCCGCGGCGGGUUAGUCGGGCCUAAGGGGAAGCGCGAGCGACACCCGAUGGACAACAGGUAGACAGUCCUGUACCGGUUAUGCGCCGUUUGACGGACGCGGGACGCUGGGGGGUAGGCGCUGCGGUCAGACGAGGACCGUCCAAGCUGGUAGGGAGACGGCGGAGGGAAGUCCGCGCCGUCGGCGACCGAGAAGCGAGAGCGAGCUGGCGCGCGAGCGCACGGCAAGAGCGUUGAACCCGCACAGCCGAGAAAGGCCUGCUACGGAGGCGCAUGACCGCCCGUACCGCAAACCGACCCAGGUAGGCUGGCUGAGGAAGCUAAGGUGGACGAGUGAUCCCUGGUUAAGGAACUCGGCAAAUUGACCCCGUACCUUCGGAAGAAGGGGUGCUCGGUCGGUGAACUCCGGCGCGGAGGGAGCCGAUGCGAGUCGCAGCAAAGCGGCCCAGGCGACUGGAUACCAGAACCACAGGUCCGUGCUGAAGCCGAAAGGCGACGUAUACGGGCUGAUGCCUGCCCAGUGCCGGAAGGUUAAGGGGAGGGCUGUAAGGUCUGAACCGAAGCCCCGGUGAACGGCGGCUGUAACUAUAACAGUCCUAAGGUAGCGAAAUUCCUUGUCGGGUAAGUUCCGACCCGCACGAAAGGCAUCACGAUCUGGGCAGUGUCUCGACCAGGGGCUCGGUGAAACUGCGCUGGCCGUUAGGACGCGGCCAACCCGUAGCAGGACAAAAAGACCCCGUGGAGCUUUACUACAGCUUGCCAUUGUGCGCGCUCCGGGCUUGCGUAGGAUAGGUGGGAGCCGGAGAACGGACCCUUGCGGGGGUGCGGGAGGCGACGGUGAAAUACCACUCUGGCACGGCGUGCGCACUCACCCGCGAGAGCGGGGACCGUGGCUGGCGGGUAGUUUGUCUGGGGCGGACGCCUCCUAAAAGGUAACGGAGGCGCGCAACGGCGCCCUCAGGCGGGAUGGCAAUCCGCCGGUGAGUGCAAAGGCAGAAGGGCGCUUGACUGCAAGGCAGACAGGCCGCGCAGAGAUGAAAGUCGGCCUUAGUGAUCCUACGGUUCCGCGUGGAAGGGCCGUAGCAUAACGGAUAAAAGCUACCCCGGGGAUAACAGGCUGAUCGUGCCCAAGAGUUCACAUCGACGGCACGGUUUGGCACCUCGAUGUCGGCUCGUCGCAUCCUGGGGCUGGAGUCGGUCCCAAGGGUUGGGCUGUUCGCCCAUUAAAGCGGCACGCGAGCUGGGUUCAGAACGUCGUGAGACAGUUCGGUCUCUAUCCGCUACGGGCGGUUAGUGGCGUGCGGGGAGCUGCUCUUAGUACGAGAGGACCGGAGUGGACGGACCGCUGGUGGAGGAGUUGUGGGGCGGCCUGCAGGCUCCGUAGCCAUGUCCGGCGGCAAGAACCGCUGAAAGCAUCUAAGUGGGAAAUGCCCCCCAAGAUGACGCCACUCACGACGGAAGGUCGGUAAGUCCCCCAGAAGACGACUGGGAAGGCGGCGCCAGCUGGAGGCCCCGUGAGGGGUAGAGGCGAGGCGUGCGCAUGGACGAGGGCUU.
?. [Build a tree for the LSU region (if available)]
Choose “LSU”.
?. [Build a tree for the LSU region (if available)]
Click “removed”.
?. [Build a tree for the LSU region (if available)]
Click next to “Search and Classify”.
?. [Build a tree for the LSU region (if available)]
Change “Min. Identity with query” to 0.7.
?. [Build a tree for the LSU region (if available)]
Click next to “Compute tree”.
?. [Build a tree for the LSU region (if available)]
Under “Output settings”.
?. [Build a tree for the LSU region (if available)]
Change “reject sequences below…” to 50.
?. [Build a tree for the LSU region (if available)]
Under “advanced tree building parameters”.
?. [Build a tree for the LSU region (if available)]
Click “include neighbors from…”.
?. [Build a tree for the LSU region (if available)]
Give the job a name and click run, the job will now show up under “Alignment Taskmanager”.
?. [Build a tree for the LSU region (if available)]
When the job is finished, click on it and click “show results”.
?. [Build a tree for the LSU region (if available)]
An “Alignment Results Table” will now appear.
?. [Build a tree for the LSU region (if available)]
Click on “Export to CSV” to save this information.
?. [Build a tree for the LSU region (if available)]
Go back to the “Alignment Taskmanager” and click on your job.
?. [Build a tree for the LSU region (if available)]
Now click on “Download file” and then “zip” to download your tree.
?. [Viewing tree using Dendroscope ]
Open the tree in Dendroscope.
See here for additional details and how to download Dendroscope: https://peerj.com/articles/960/#building-a-16s-rdna-treeFigTree (another tree viewing software) also works!
?. [Viewing tree using Dendroscope ]
Re-root the tree to the outgroup.
?. [Viewing tree using Dendroscope ]
Change the view to “phylogram" to make it easier to look at.
?. [Viewing tree using Dendroscope ]
Write down who is the closest sister taxa/group and check to see who that taxa/group is. Now assess your phylogeny.
Where do your sequences fall on the tree? Does this make sense with your BLAST/RDP assignments?
?. [Build phylogeny manually]
Download fungal sequences to include in the tree from BLAST and the literature.
?. [Build phylogeny manually]
Based on the fungal taxonomy of your sequences, decide which of your fungi to group into a single tree.
e.g. all the fungi that blast to Penicillium or all fungi in the same “Genus” or “Family” based on RDP.
?. [Build phylogeny manually]
Next write down the accession numbers for fungal sequences from BLAST that are close matches.
e.g. the top hits on BLAST - maybe take the top 3 hits for each taxa. As you make your list for all the top hits to your taxa, you may have duplicates if many of your fungal taxa are similar, but we don't want duplicate sequences in our tree so you can remove the duplicate accession numbers from your list now.
?. [Build phylogeny manually]
Identify an outgroup and also some close relatives from other closely related genera (this is the ‘hardest’ part).You can do this through literature searches (e.g. Google Scholar, “Fusarium phylogeny”) and writing down                                   accession numbers used to build trees previously. Or you can use BLAST/GenBank Taxonomy Viewer to pick some fungal taxa. For example:1. Click on the Accession number of a top hit for one of your sequences.2. Click on the “Organism”.4. Check “nucleotide”. 5. You can also click on other levels in the “Lineage”. e.g. “Family” (Debaryomyces, etc) to obtain close relatives outside of the group of interest.6. If you now want to obtain the Accession numbers of these fungal taxa to use in your tree, (in this example we are going for a sequence in the same Genus (Candida), but that is not Candida saitona).      a. Click on the name of the Genus you are interested in.      b. Then on the right, click on the number by “Nucleotide”, this will take us to a list of all the DNA sequences for this organism.      c. At this point, you may see a sequence already that says “large subunit ribosomal RNA gene”, this what we want, if not, you can search for this - just paste “large subunit ribosomal RNA gene” after what is already included in the search bar.      d. Ideally you should then see a hit that looks like this!! Write down that Accession Number.
These are all fungi where there is available DNA data in the database (the number next to the taxa is the number of DNA sequences available).
When building a tree you want to have:                i. 2-3 very close relatives (obtained previously based on ‘top hits’).                ii. And then 2-3 sequences from the same Genus (e.g. Candida) that are not the closest hits (so not from the same species (so in our example, not Candida saitoana)).                                                       iii. 2-3 sequences that are outside the Genus (so from other Genera in the Debaryomyces).                iv. 1-2 sequences that are even more distant (so from another Order of fungi, etc).
?. [Build phylogeny manually]
You should now have a list of Accession numbers that looks like this - with a new Accession number on each line - save this as a text file (e.g. .txt). We now want to obtain the sequences for all of these.Go to https://www.ncbi.nlm.nih.gov/sites/batchentrez.“Database” should be “Nucleotide”.Select your file of Accession numbers.Click “Retrieve”.It will then take you to a screen where it tells you if it had any issues with your Accession numbers (e.g. duplicates, etc), and here you want to click on “Retrieve records”. 5. This will take you back to NCBI which will now list all the sequences you want, you now want to find “Send to” on the right of the screen and pick “File”, Format of “Fasta”.6. Yay, you’ve just downloaded all the sequences! Now you just need to combine these sequences into one file with your sequences (so copy + paste into one file). You should now have one “fasta” file that contains all your fungal sequences and all of the close relatives and outgroup sequences obtained from the above methods.
?. [Build phylogeny manually]
Generating an alignment.
?. [Build phylogeny manually]
We now need to align these sequences so that we know how each of the nucleotides in each sequence lines up.Go to CIPRES (https://www.phylo.org/portal2/home.action) and create an account.
?. [Build phylogeny manually]
Log in to CIPRES. (https://www.phylo.org/portal2/home.action) and create an account.
?. [Build phylogeny manually]
Make a new project folder (e.g. Test).This folder has both “Data” and a “Tasks” folders inside.
?. [Build phylogeny manually]
Click on “Data”.
?. [Build phylogeny manually]
Upload your fasta file with all your fungal sequences.
?. [Build phylogeny manually]
Now click on “Tasks".
?. [Build phylogeny manually]
You want to create a new “Task”.
?. [Build phylogeny manually]
Enter something that makes sense in the “Description” to name the task, eg. Fungal alignment with MAFFT.
?. [Build phylogeny manually]
Next click “Select Input Data” and pick your uploaded fasta file.
?. [Build phylogeny manually]
Next “Select tool” and pick MAFFT (alignment software).You should now have something that looks like this:
?. [Build phylogeny manually]
Click “Save and Run Task”.
?. [Build phylogeny manually]
You will get an email when it is finished running and then you can go back to CIPRES, to “Tasks” and now click on “View Output".
?. [Build phylogeny manually]
You want to click “Download” next to the “output.mafft” file.You now have your aligned sequences!
?. [Build phylogeny manually]
Trim the alignment (remove parts that don’t overlap).
?. [Build phylogeny manually]
Go to http://phylemon2.bioinfo.cipf.es/.
?. [Build phylogeny manually]
Sign in / make an account or work as an “anonymous user”.
?. [Build phylogeny manually]
Go to “Utilities”.
?. [Build phylogeny manually]
Click on “Alignment Utilities”.
?. [Build phylogeny manually]
Click on “TrimAl”.i. Upload your aligned sequence file from CIPRES.ii. Now edit the parameters for TrimAl, click on the checkmark by “delete spurious ….” and add the values 0.75 and 50 respectively (or for advanced users, your values of choice) and then also select “gappyout”.iii. Now run the job!.iv. When its finished running you want to download the new fasta file, just click on “outfile.out” to download.
If its running, it should show up in the job list!
?. [Build phylogeny manually]
Now we want to look at our alignment! (optional, but recommended).
?. [Build phylogeny manually]
Download JalView (http://www.jalview.org/).
?. [Build phylogeny manually]
Open JalView and drag your trimmed alignment file into JalView and it should open it up.
For beginners - you just want to make sure that it looks like things “line up” and that nothing looks too “weird”.
?. [Build phylogeny manually]
Next, we can either make a slower, potentially more accurate phylogeny or a faster, potentially less accurate phylogeny. To make a slow phylogeny skip to steps 32 and 33. To make a fast phylogeny go to step 31.
?. [Build phylogeny manually]
Build a phylogeny using FastTree2.
?. [Build phylogeny manually]
Go back to CIPRES.
?. [Build phylogeny manually]
Click on "Data".
?. [Build phylogeny manually]
Upload your fasta file (outfile.out) with your aligned trimmed sequences.
?. [Build phylogeny manually]
Now click on "Tasks".
?. [Build phylogeny manually]
Create a new "Task".
?. [Build phylogeny manually]
Enter something in the description (e.g. fasttree of Candida).
?. [Build phylogeny manually]
Download the file that ends in ".tre".
Yay we have a tree!. You can stop here unless you want to make a more accurate tree in steps 31 and 32 below.
?. [Build phylogeny manually]
When finished “View output”.
?. [Build phylogeny manually]
Save parameters and submit task.
?. [Build phylogeny manually]
Change “Maximum Hours to Run (up to 168 hours)” to 2.
?. [Build phylogeny manually]
Click on “Parameters”.
?. [Build phylogeny manually]
Next "select tool" and pick FastTreeMP on XSEDE.
?. [Build phylogeny manually]
Next "Select input data" and select your uploaded file.
?. [Build phylogeny manually]
Pick an evolutionary model!.
?. [Build phylogeny manually]
Back to CIPRES!.
?. [Build phylogeny manually]
Next “Select tool” and pick JmodelTest2.
?. [Build phylogeny manually]
Next click “Select Input Data” and pick your uploaded file.
?. [Build phylogeny manually]
Enter something that makes sense in the “Description” to name the task, eg. Fungal model test.
?. [Build phylogeny manually]
You want to create a new “Task”.
?. [Build phylogeny manually]
Now click on “Tasks”.
?. [Build phylogeny manually]
Upload your fasta file (outfile.out) with all your trimmed and aligned sequences.
?. [Build phylogeny manually]
Click on “Data”.
?. [Build phylogeny manually]
Build a phylogeny !! Finally!!
?. [Build phylogeny manually]
Click on “Parameters”.i. Change “Maximum Hours to Run (up to 168 hours)” to 2.ii. Under “Configure Model Search” set the number of substitution schemes to 3.
?. [Build phylogeny manually]
Save parameters and submit task.
?. [Build phylogeny manually]
When finished “View output”.
?. [Build phylogeny manually]
Also on CIPRES!.
?. [Build phylogeny manually]
Next click on “View” next to STDOUT.
?. [Build phylogeny manually]
Scroll to the bottom and find this information:
This tells us which evolutionary model works best, hopefully they all match! Very likely it will say GTR + I + G or GTR.
?. [Build phylogeny manually]
Now click on “Tasks”.
?. [Build phylogeny manually]
You want to create a new “Task”.
?. [Build phylogeny manually]
Enter something that makes sense in the “Description” to name the task, eg. Fungal phylogeny.
?. [Build phylogeny manually]
Next click “Select Input Data” and pick the same file you just used for the model test above (the trimmed and aligned file).
?. [Build phylogeny manually]
Next “Select tool” and pick MrBayes. Note for advanced users: CIPRES also supports RAxML
?. [Build phylogeny manually]
Click on “Parameters”.
?. [Build phylogeny manually]
Pick “MrBayes 3.2.6”.
Now we want to set up the evolutionary model we decided on above (see: https://gist.github.com/brantfaircloth/895282).E.g. for GTR + I + G
?. [Build phylogeny manually]
Save parameters and submit task.
?. [Build phylogeny manually]
You will get an email when it is finished running and then you can go back to CIPRES, to “Tasks” and now click on “View Output”.
?. [Build phylogeny manually]
Download the “infile.nex.con.tre” file and open this file in a tree viewing software to see your tree!.
YAY! We finished making our tree!.
?. [Viewing tree using Dendroscope ]
Expand the tree by clicking the expansion button, then scroll through the tree to locate the outgroup.
?. [Viewing tree using Dendroscope ]
Click on the beginning of the taxon name, to select it, and reroot the tree by going to edit and selecting “re-root”.