By Allan Gonzalez
These are some links that helped me study for the certification test:
– Youtube video that helps you understand what you will be tested broadly.
– Reddit thread that guided me.
– Online practice test.
Home» Articles posted by jirihulcr
Articles posted by jirihulcr
Shipping beetles to the UF Forest Entomology Lab
Thank you for allowing us to take a look at your beetles! Rest assured that they will be treated professionally.
If you are sending them to be identified for your research, please make sure you provide this information.
If you are depositing the beetles into the UF Forest Entomology collection, here is how they may be used :
– If your samples are of high quality (preserved in high-concentration, non-denatured ethanol from the moment of beetle death, and refrigerated) they will be deposited in -80C and in 99% ethanol, which will preserve them for molecular analyses of the beetles or the fungi vectored by the beetles. They may be used by our team or by anyone else in the global bark beetle research community.
– If your samples are of lesser quality but still morphologically intact, they will be used teaching, such as in the Bark Beetle Academy.
Packaging
1) Please put your specimens in ethanol in a plastic, screw-top vial. Do not use snap-top vials (sometimes called Eppendorf tubes) because they invariably pop open in high altitudes and leak. Do not use glass vials – they break.
2) Add a piece of soft napkin or cotton; this prevents the beetles from trashing around and breaking their legs and antennae.
3) The ethanol is important to dehydrate, clean and preserve the specimens. However it is flammable, so you can’t ship it legally. So after the specimens soak and dehydrate, we recommend pouring off about 90% of the ethanol so you can declare the shipment “dry”.
4) Place your vials on a sheet of paper and attach them with a sticky tape.
5) Put the whole thing into a padded envelope.
Shipping
You can send it cheap regular post. Or feel free to use our Fedex account, so you don’t have to pay anything (happily available on request).
Our lab address is:
Jiri Hulcr
UF School of Forest Resources and Conservation
1745 McCarty Drive
Gainesville, FL 32611
USA
Phone: 517-256-1894
Permits
If you are shipping beetles form overseas, we strongly recommend that you attach a document, confirmed by your institution, stating that the beetles were collected with the appropriate permits issued by the country of origin, and that the sample is provided for mutually beneficial research. (Obtaining permits for dead, dry, common pests should not be difficult.) On the US side, you do NOT need a permit to send such specimens into the us (US regulation 7CFR 330.200).
THANK YOU!
Geneious
We have the latest version of Geneious R9. To use it on one of the three lab computers, everyone must enter the license information (listed below) when prompted upon opening it for the first time under your user profile. This needs to be done on each of the three lab computers – entering the key into one computer does not grant automatic access on the other. After that point you should be good, but let me know if that is not the case. For the “Licensee”, you must enter Jiri’s name as shown below, not yours.
Please DO NOT download the new version of the software from the Geneious website and use it on any other computer. Our license allows for three computers to have it installed on.
Our License Information
1 x NON-COMMERCIAL PERSONAL LICENSE
Licensee: Jiri Hulcr
Importing live fungi
These are the conditions on our USDA permit for hand-carrying live fungi through airport customs. All fungi have to be on minislants.
1. At least TWENTY days prior to each hand carry incident, the permit holder or designee must notify the PPQ Permit Compliance Officer by email (redandwhitelabelrequest@aphis.usda.gov) to provide specific information on the hand carrier’s identity, the anticipated first port of arrival into the United States, the actual date of arrival, the time, and, if travel is by airline, the flight number. The Compliance Officer will notify Customs and Border Protection (CBP) Agriculture Specialist at the port of entry to document and facilitate the entry of the organisms.
2.The hand carrier must indicate that living organisms are being imported under a USDA permit on the Customs Declaration form if such form is required at the port of entry.
3.At the port of entry, individuals carrying permitted organism must also present to CBP officers the following articles: Passport or Visa and a valid hand carry PPQ Form 599 Red/White label corresponding to the permit.
4.Inspection by CBP Officers must confirm that all hand carried articles are securely packaged as per the permit conditions. In the event that a problem is detected, the CBP officer may seize the package and require its movement to the nearest PPQ Inspection Station for processing, clearance or destruction. The permit holder will be responsible for all costs incidental to such forwarding.
5.After CBP confirmation and clearance through the first port of entry into the United States, hand carried organisms must be transported directly to the containment facility authorized in the permit.
6.Upon arrival at the facility, the PPQ Compliance Officer must be notified within 24 hours that the organisms arrived. Notification may be by fax (301-734-5392) or email (redandwhitelabelrequest@aphis.usda.gov). Notification must be by an independent third party (e.g. containment facility director, departmental chair, campus biosafety officer, etc.). The notification must include the permit number, label number, date of arrival, the specific organisms that were imported, their origin, and quantity. Failure to notify the PPQ compliance officer may result in loss of hand carry privileges. A PPQ inspector may also visit the facility to confirm the arrival of the package and its contents.
7. ONLY PERSON(S) WHOSE NAME(S) IS/ARE LISTED IN THE ISSUED PERMIT IS/ARE AUTHORIZED TO HAND CARRY.
8. You will receive NEW PPQ Form 599 Red/White labels for each hand carry event once you have submitted the required information. You can NOT use the red and white labels described above that are prepared for BONDED CARRIERS. If you use the PPQ Form 599 Red/White labels for bonded carrier while attempting to hand carry, the package will be seized by the Department of Homeland Security and destroyed.
9. An authorization to hand-carry includes only the organism identified in the permit. Presence of unauthorized organisms in any packages on an individual authorized to hand-carry is a permit violation. Presence of unauthorized organism at the receiving containment facility at any time is also evidence of a permit violation.
Website management
Our ambrosiasymbiosis.org website is a part of the backyardbarkbeetles.org site on BlueHost. Log in as backyay3. To edit a page, go to File Manager, open the folder public_html. Editing directly online is easiest.
How to view various website statistics:
For the Emerging Threats webpage, visit webstats.ifas.ufl.edu. Use the log in credentials below:
User: sfrc.ifas.ufl.edu
Pass: ifasstats
Then, select Content Optimization > Navigational Analysis > All Navigation and find the /emergingthreats/ subfolder. The navigational analysis for the selected date range will appear on the right.
Primers for MiSeq amplicon sequencing
General construction of double-indexed fusion primers for pair-end sequencing on MiSeq:
P5 (adapter) linker index i5 SBS3 (sequencing primer, read 1) template primer fwd AATGATACGGCGACCACCGA GATCT xxxxxxx ACACTCTTTCCCTACACGACGCTCTTCCGATCT ZZZZZZZZZZZZZZZZZZZZZZZ
P7 linker index i7 SBS12 (sequencing primer, read 2) templ primer reverse
CAAGCAGAAGACGGCATACGA GAT xxxxxxx GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT YYYYYYYYYYYYYYYYYYYY
This construction is based on the TruSeq chemistry. At ICBR, they know it but it might be worth emphasizing when you submit your libraries. For more details on preparing a library using these fusion primers go here.
The steps of pair-end sequencing are as follows:
- SBS3 priming, read 1 sequencing
- i5 sequencing
- i7 sequencing
- SBS12 priming, read 2 sequencing
Therefore, the i7 index is the one that’s read first. We already have 96 versions of the read 2 primer, the one with the i7 index. The i5 index can multiply your multiplexing power.
Issues with base diversity
Illumina machines have trouble detecting signal from individual DNA clusters if all/most clusters have the same base across the lane. This is especially problematic with amplicons, where most sites will have the same letter in most reads. It can result in unclear signal. That is important to avoid especially in the indexes and at the beginning of the read when the instrument is still calibrating itself. There are two strategies to avoid low-quality reads due to homogeneity of DNA:
- Spiking with PhiX (a phage genome) – ICBR does that automatically, but you need to tell them that you have an amplicon, so they add more. That works, but it wastes sequencing power.
- Make the sequences mutually off-set, either by adding heterogeneity spacers, or by using sliding template primers. We use the latter. It is possible is the surrounding of the priming site is also fairly conserved. An example of a sliding LR0R primer (fungal LSU) is here:
priming site on template: 5'...GCCACCCGCTGAACTTAAGCATAT..3' original LR0R: ACCCGCTGAACTTAAGC LR0R(1): CCCGCTGAACTTAAGCA
The first PCR (to enrich for your fragment) should be done with the same uniform template primer, but these sliding primers can be incorporates into the fusion primers. They assure that the reads are off-set by one position which creates the desired base diversity at every sequencing step.
It is also very important to make sure that indexes re ballanced
Issues with read length
Our primers
Here are ALL the barcodes/indexes you can choose from. Although the combination of two indexes should unambiguously identify the original sample, it is not a bad idea to use unique indexes throughout, both for the i5 and i7 segments:
MiSeq library prep for fungus community metabarcoding
Nested PCR
- The initial DNA extract template is subjected to amplification with the template-specific primers LR0R and JHY-LSU-369rc for 30 cycles. The number of cycles is lower than usual to prevent various biases resulting from excessive amplification.
- PCR reactions are not cleaned up, to avoid loss of DNA (we used to do it, but it is probably not necessary).
- 5μl of each amplicon is run on a gel to assess failures/successes. You may not see very much on your gel! Even faint band should be used. When band is invisible, I wouldn’t use it even though there may still be some amplicon – it was probably not a good unbiased amplification.
- A 1-μl aliquot of each successful amplicon is used as a subsequent PCR template and subjected to a 20-cycle amplification with the fusion primers.
- The secondary (final) amplicons are cleaned up with AmPure DNA cleanup kit.
- The DNA concentration in each cleaned PCR product is measured using the PicoGreen-based Quant-iT kit.
- Products are pooled in equimolar proportions to create an amplicon library at a final concentration of 10 ng μl−1.
- The pooled amplicon library is cleaned up using UltraClean PCR clean-up kit and submitted to ICBR.
- When submitting to ICBR, make sure you specify that we need raw output, non-demultiplexed fastq data.
The LSU fusion primers
LR0R –> JH-LSU-369rc: From 5’ of the first primer site to the 3’ end of the reverse primer: 400bp. The most variable region (positions): 100-240
Primer construction
To prepare an order, simply concatenate. You need one Forward, and as many Reverse as you need barcodes. We order form IDT, which only does synthesis at 100nM for oligos this long. I have negotiated this synthesis for the price of 25nM synthesis, so do purchases through me. Specify that you want them in the “Lab Ready” suspension.
Forward primer:
P5 AATGATACGGCGACCACCGA
Linker GATCT
SBS3 ACACTCTTTCCCTACACGACGCTCTTCCGATCT
LR0R ACCCGCTGAACTTAAGC
Reverse primer:
P7 CAAGCAGAAGACGGCATACGA
Linker GAT
Barcode xxxxxxx
SBS12 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
JH-LS-369rc CTTCCCTTTCAACAATTTCAC
Barcodes:
We already have the JH-LS-369rc fusion primers with these barcodes :
1 AAGGTGG
8 GCTGAGG
15 ACAGCGG
23 CATGGTG
31 TCGGTTG
35 CGAGATG
41 TGTGCTG
45 CTAGGAG
51 TATGTAG
56 GTGGAAG
60 TAATCAG
64 TCTGGCG
70 CCAGTCG
75 CTGTACG
78 GATGCCG
80 ACGTGGT
85 TCTGTGT
93 TAAGAGT
98 GTTGCGT
103 ATTGGTT
109 CTGGATT
114 TAGGCTT
120 GGTGGAT
127 CAAGTAT
134 TCGTAAT
138 CCGGCAT
142 AATTGCT
146 ACGGTCT
150 GAGGACT
155 TTAGCCT
158 CATTGGA
167 GATCTGA
176 GCTGGTA
181 AGTGACA
189 TTCAGGC
202 GACGGTC
Other available barcodes:
2 CTTGTGG
3 TGAGTGG
4 CAATTGG
5 TTCTTGG
6 GGTATGG
7 GTGCTGG
9 ATCGAGG
10 GAGTAGG
11 TGTTAGG
12 AGGAAGG
13 TCGCAGG
14 AATCAGG
16 TACGCGG
17 ATGTCGG
18 GGATCGG
19 CAGACGG
20 GTCACGG
21 CGTCCGG
22 TTACCGG
24 TAGTGTG
25 AGATGTG
26 CTCTGTG
27 ACTAGTG
28 TGCAGTG
29 ATGCGTG
30 GCACGTG
32 GTAGTTG
33 AGCGTTG
34 CTGATTG
36 ACGTATG
37 TTATATG
38 GTTAATG
39 AACAATG
40 CAGCATG
42 GCCGCTG
43 AATTCTG
44 CCATCTG
46 CCTTGAG
47 GGAAGAG
48 CACAGAG
49 TGGCGAG
50 GATCGAG
52 GCGTTAG
53 ATATTAG
54 TCCATAG
55 AGTCTAG
57 TGCGAAG
58 CTCCAAG
59 ATTGCAG
61 AGCTCAG
62 GCTACAG
63 AAGCCAG
65 GTCGGCG
66 AAGAGCG
67 CTTAGCG
68 CCGCGCG
69 TAACGCG
71 TGGTTCG
72 AGAATCG
73 CATCTCG
74 ATCCTCG
76 GCATACG
77 GGCAACG
79 TCGACCG
81 CTATGGT
82 CGTAGGT
83 GACAGGT
84 AGACGGT
86 ATAGTGT
87 TAGTTGT
88 GCATTGT
89 AGCTTGT
90 CCGATGT
91 AATATGT
92 CTCCTGT
94 CACTAGT
95 GTGAAGT
96 TCCAAGT
97 CGGCAGT
99 CGAGCGT
100 CCTTCGT
101 TGGACGT
102 GAGCCGT
104 TCAGGTT
105 GTGTGTT
106 AGGAGTT
107 CCTCGTT
108 TACCGTT
110 GGCGATT
111 GCTTATT
112 TATAATT
113 AGTCATT
115 CGGTCTT
116 ATCTCTT
117 GCGACTT
118 CTTACTT
119 GTACCTT
121 AACGGAT
122 GAATGAT
123 CGCTGAT
124 TTGAGAT
125 ACAAGAT
126 GCGCGAT
128 GCCGTAT
129 CTGTTAT
130 TGATTAT
131 GAGATAT
132 GTTCTAT
133 AGCAGGA
135 GTCTAAT
136 CATCAAT
137 GGACAAT
139 AGTACAT
140 TACACAT
141 TCTCCAT
143 TTCTGCT
144 GCTAGCT
145 TGAAGCT
147 CGTGTCT
148 TCACTCT
149 GGCCTCT
151 AGATACT
152 CTCAACT
153 TTGCACT
154 AACCACT
156 GGTTCCT
157 ATGACCT
159 GCCTGGA
160 CTGAGGA
161 TAGCGGA
162 ACTCGGA
163 GTACGGA
164 CGCGTGA
165 TTAATGA
166 AGGCTGA
168 CCACTGA
169 CTAGAGA
170 GACGAGA
171 CCGTAGA
172 TTCCAGA
173 AATGCGA
174 TGCTCGA
175 TCTACGA
177 TCGAGTA
178 GTCAGTA
179 CGGCGTA
180 ACAGTTA
182 TATTACA
183 CGGAACA
184 CTTCACA
185 CTGGCCA
186 ACCGCCA
187 TCATCCA
188 TTCACCA
190 GTCGTGC
191 CAGCTGC
192 TGTCTGC
193 ACGGAGC
194 CGTGAGC
195 TTGTAGC
196 GGCTAGC
197 GTTCAGC
198 AGCGCGC
199 GATTCGC
200 CTCTCGC
201 GCACCGC
203 ACCTGTC
204 GGTAGTC
205 AATCGTC
Bench adaptation to microscope
Adam,
Whenever you have a moment, will you be able to try to remove one of the unnecessary bench parts that are preventing people from sitting down with a microscope?
Thanks!
AmPure magnetic DNA clean up
Adam,
We bought the Ampure DNA purification system. Would you be able to put it together, explore how it works, and give us training?
It might be worth taking a PCR product that is no longer needed but had a strong band (something that we already sequenced) and runningon a gel 10 5ul of the original, 2) 5ul Ampure cleaned-up version, and 3) 5ul MoBio cleaned version.
The sooner the better (within two or three weeks?), because I am imagining that Craig and possibly Tyler Dreaden might want to use it for the next MiSeq library.
For individual Sanger sequencing submission, we will still be using ExoSap. This is only for large volumes that have to be really clean, like whole library submissions.
Thank you!
J
anobiid photo
Adam, in “your box” in my office is a sample of wood. Could you please take a photo of the bark side (female butts visible – please be careful, they are dead, old and fragile!) and of the internal winding frass-filled tunnels? I would like to show you in person first… This is for a book, you will be included as the photographer.
