Frequently asked questions (FAQ)
- FST Production
- What is the genetic background of GABI-Kat lines?
- Which vectors have been used to create which lines?
- Where can I get more information about vectors and primers used at GABI-Kat?
- What is the selection scheme?
- How were the flanking sequences generated?
- Database Search
- What do I need to know to use SimpleSearch?
- How can I evaluate the sequence which defines the hit?
- How are the FST sequences processed?
- How is a "gene hit", a "promoter hit", a "TS2TE hit" and a "CDSi hit" defined at GABI-Kat?
- I found several FSTs indicating an insertion at the same position, and they correspond to very similar line IDs, like 012B03, 012B04 and 012C04. Which one should I choose?
- Is the insertion site of the FST in the Graphic View exact?
- Which genome annotation is the basis for the current version of SimpleSearch?
- What does the "line availability" on the show FST page mean?
- What does it mean if SimpleSearch reports the "Confirmation Status" of an insertion to be "failed"?
- Seed Ordering
- How long it usually takes to receive the ordered lines?
- Why do we sometimes receive seeds very long after an order?
- What is the confirmation rate?
- How can I check the status of my seed order?
- Could you tell me if GK line 123A04 has been ordered by someone else before, and who?
- I have received the seeds with a report sheet. Can I get the sequences on the report sheet electronically?
- The new MTA (pv20050624) with a line fee of 100,- EUR is available on your web site since 24.06.2005. What happens with line orders which were placed before that date?
- I am interested in getting GABI-Kat lines, but how do I know if and when these lines will be available at NASC?
- If I order a line from GABI-Kat and confirmation is successful, when is that line given to NASC?
- What happens to the executed MTAs that are sent to GABI-Kat?
- I ordered lines from GABI-Kat before, and now I would like to order a few new lines. Do I need to send a new MTA for a new order?
- Do you accept payment by credit card?
- What happens if my institution does not pay the line fee?
- What does the name GABI-Kat stand for?
- How is the term "T1" interpreted by GABI-Kat?
- How to get a probe to check for the insertion on a DNA gel-blot?
- What is the difference between a single line and a T3-set at NASC?
- What if my question is not answered in the FAQ?
- I wrote e-mail to GABI-Kat and I always got reply e-mails with something like GKT:XXXXXXXXXX or CBT:XXXXXXXXXX in the subject line, what's that?
- How should I cite GABI-Kat in my publication?
- How to identify a second insertion in a GABI line?
- Why do I get seeds which I ordered a very long time ago?
- Are ALL GABI-Kat lines sul resistant?
- What is the genetic background of GABI-Kat lines? Columbia-0
- Which vectors have been used to create which lines? We have used four vectors: pAC106 (GenBank: AJ537513), pAC161 (GenBank: AJ537514), pGABI1 (GenBank: AY529716) and pADIS1 (GenBank: AY529717). Sequence and overview map data of all vectors are available from the download page. Features of interest which are not included in the map should be deduced from the sequence. For a specified line, the vector is displayed in the "Show Sequence" page of SimpleSearch. The link between block number and vector is as follows (block number is the first 3 digits of the line ID and '-' is inclusive):
blocks 001-032, 801-849 => pAC106
blocks 500, 566, 567, 579, 580, 587, 588, 597, 645, 654, 658-660, 662, 664, 666, 692, 693, 699, 700, 702-739, 741, 748, 763, 764, 774-776 => pGABI1
block 568 => pADIS1
all other blocks => pAC161
- Where can I get more information about vectors and primers used in GABI-Kat? You can get more information about vectors and primers in the vector and primer info page.
- What is the selection scheme? The answer is on the sul selection scheme page.
- How was the flanking sequences generated? Please see the sequencing strategy page. More details can be found in the paper: Strizhov N et al. BioTechniques 2003 35:1164-8. [PubMed]
- What do I need to know to use SimpleSearch? To use GABI-Kat SimpleSearch, you need to know the A. thaliana AGI gene code (e.g. At3g55120 for chalcone flavanone isomerase) for any AtYFG (Your Favourite Gene from A. thaliana). You can use TAIR or MAtDB at MIPS to find these codes for any annotated AtYFG. There is also a sequence-based (BLAST) search option integrated into SimpleSearch; you can search with the DNA or protein sequence from AtYFG.
Note: see FAQ 12 for the genome annotation version we are using.
- How can I evaluate the sequence which defines the hit? First, you need to check which primer was used to generate the respective sequence. This information is encoded in the designation of the sequence: the last field contains the primer name (e.g. 8409 in 65-K012471-022-054-A09-8409). Many, but not all, sequences were generated with primer 8409 which reads out of the left border (LB). A list of all primers and the respective sequences is available from the GABI-Kat sequencing strategy page. Second, use the FST sequence to confirm our automatic annotation of the FST which is shown in the field "Predicted Position of Insertion" (example for 051G10/AT4G23270: Chr4:12173256). From the FST sequence, you can deduce the approximate insertion location.
Please note that the insertion position deduced from FSTs is not absolutely reliable, especially when no T-DNA sequence is detected at the beginning the FST (and also depending on the quality of the respective sequence read).
- How are the FST sequences processed? One of the first steps of FST processing is T-DNA vector sequence detection and the subsequent removal (masking) of these sequences. For the re-annotation of all our FST in 2010 (data released February 2011 with v24 of the SimpleSearch database), we have used the distance of the sequencing primer annealing site to the BLAST match to predict the insertion site. This distance information was deduced from the original trace files.
- How is a "gene hit", a "promoter hit", a "TS2TE hit" and a "CDSi hit" defined at GABI-Kat? Since we use the A. thaliana AGI gene code (see FAQ 12) as a basis for searching and identifying insertions, we use the terms "genome hit", "gene hit", "TS2TE hit" , "promoter hit" and "CDSi hit" to describe the quality of FST's and insertion alleles. The classification is generated by transferring the information of the TAIRv10 annotation at the predicted insertion position to the predicted hit. The predicted insertion position is presented for each FST as a pseudochromosome coordinate. An insertion is
- a genome hit, when the sequence shows significant similarity to the A. thaliana genome sequence. The locus of a genome hit is described by the ID of the annotation unit (BAC-ID) of the respective genome region (example: 048E04/F21P8);
- a gene hit, when it is a genome hit and the predicted insertion position is located between 300 bp upstream of ATG and 300 bp downstram of stop codon of an annotated gene (IF there are no UTRs annotated);
- a TS2TE hit (for "transcription start to transcript end"), when it is a genome hit and the predicted insertion position is located between transcription start and transcript end of an annotated gene (IF UTRs are annotated);
- a promoter hit, when it is a genome hit and the predicted insertion position is located between 300 bp upstream of the transcription start (IF UTRs are annotated; we use the 5' end of the UTR as the position for TS);
- a CDSi hit, when it is a genome hit and the predicted insertion site is located between ATG and STOP in genomic DNA (CDS plus introns) of an annotated gene. Note that the gene needs to code for a protein in order to be classified as CDSi hit. In other cases, e.g. for RNA-coding genes, it "stays" a TS2TE-Hit.
Before 2011-02-15, we used a less exact definition of gene hit: the FST overlaps with the genomic sequence between ATG and STOP (CDS plus introns) of an annotated gene. The classification "TS2TE" has been added during the re-annotation of all our FSTs in 2010 (data released February 2011 with v24 of the SimpleSearch database). The change allows a more reliable selection of insertion alleles that may be NULL alleles. For a short time between February and July 2011, we used the term "TS2pA". However, this was replaced by TS2TE to include RNA-coding genes that have no polyA tail at their transcripts. Note that we refer with TE to the "transcript end" of the mature transcript without polyA that is usually found in the cytosol, not "transcription end" of the primary transcript.
The change in the definition has an effect on the hit rate. The reason is that the insertion predictions show a preference for the gene area close to TS and TE, and that the size of the genome area is different for the regions covered by the definitions:
(i) area for all protein coding genes from -300_ATG through CDSi to STOP_+300: 67.211.634 bp
(ii) area for all protein coding genes from TS to TE (plus (i) for all genes with no UTR): 64.184.741 bp
- I found several FSTs indicating an insertion at the same position, and they correspond to very similar line IDs, like 012B03, 012B04 and 012C04. Which one should I choose? Most probably this is because of contaminations that happen during the high-throughput PCR procedure. We STRONGLY suggest you request all of them. Finally we will confirm only one of the lines in such a "contamination group". You will be charged only for the confirmed line. There is no way to predict which line contains the true hit from the sequences (e.g. BLAST e value) in advance, because the FST from the real line could have a bad sequence for many different reasons.
SimpleSearch tells you the confirmed line (allele) when we were able to solve the respective contamination group. As an example, search for line 285D07, and check the field "Confirmation Status". The allele has been confirmed in line 285E06.
- Is the insertion site of the FST in the Graphic View exact? No, the position of FSTs in the Graphic View page is not exact. For example, the indicated positions are shifted to prevent one FST from overlapping to another. The shifted distance can be up to several hundred basepairs.
- Which genome annotation is the basis for the current version of SimpleSearch? Our current FST annotation is based on the TAIRv10 A. thaliana genome sequence and pseudochromosome annotation. Until February 2011, SimpleSearch was using TIGRv5, with release 24 we switched to TAIRv10 (see also the SimpleSearch database version information).
- What does the "line availability" on the "show sequence" page of SimpleSearch mean? If the line availability is "donated to NASC" or "available from NASC (......)", the line has been donated to NASC and can not be ordered from us; please order it from NASC (see also FAQ 22). If the line availability shows "line died - no seeds available", the line is lost and should not be ordered. Reasons for losing lines are for example sterility of the T1 plant, failed germination of T2 seed, or other reasons that result in lack of offspring seed. Note that even for lines that currently display "available from GABI-Kat" for line availability, it is still possible that for example the T2 seeds do not germinate or that all T2 plants die prior to seed set. In addition, line availability does not guaranty success of confirmation, although it is a first step in this direction.
- What does it mean if SimpleSearch reports the "Confirmation Status" of an insertion to be "failed"? We only deliver seeds of lines if the respective insertion site has been confirmed. This confirmation is successful for about 80 to 85% of the insertions. In the remaining about 20% of cases, the confirmation status of that insertion is set to "failed", and the line is not delivered. During the confirmation process, we check for DNA quality of the T2 DNA, perform a control PCR to detect the resistance gene, order a second primer when the first fails, and redo the confirmation if there are hints for technical problems. See also FAQ 16 and FAQ 17.
- How long it usually takes to receive the ordered lines? Usually it takes about three months to finish the confirmation and to send you the seed (if confirmation was positive). Some lines have only set few T2 seed, or they show only very low germination rates. In these and other related cases, we can not use T2 seedlings to prepare genomic DNA and have to wait for adult T2 plants to harvest leaves for preparing DNA. And if the T2 seeds were used up in the confirmation, we have to wait for the harvest of T3 seeds to get something we can deliver. In such a case, you will find the status "confirmed" in request tracking, but still "in preparation for sendout" for quite some time.
- Why do we sometimes receive seeds very long after an order? We try to get the confirmed lines to users within three months. In some cases, it takes longer, for example when the plants grow poorly or if the controls indicate a problem that causes a repetition of the confirmation process. However, our rule is that we do not charge the line fee if we fail to deliver within 6 months. In most cases, there is no chance to confirm a line after we attempted for such a long time. But there are exceptions, which include the resolution of "contaminations" (see FAQ 10) or errors in the assignment of line ID to FST (see FAQ 17). In these cases, it happens that even after two years seed is delivered. This should also make transparent to the person who initially ordered a line that "his" or "her" mutant is donated to NASC.
- What is the confirmation rate? The confirmation rate is about 80 % of all FSTs. The original FSTs were derived from DNA extracted from leaves of T1 plants, the confirmation is usually performed with with DNA extracted from several T2 plantlets.
Between 2009 and 2011, we have been able to push the confirmation frequency towards 85%. This was possible by resolving "logistic errors" that caused a loss of the link between the FST ID and the line ID. Such errors happen for example when a tray with plants in the greenhouse is skewed (turned around). See also FAQ 16.
- How can I check the status of my seed order? After we received all the necessary documents of your seed order, you will receive a notification e-mail which confirms that we processed your order. There is a request UID in that e-mail, and you can use this request UID to check the status of your order in the request tracking page.
- Could you tell me if GK line 123A04 has been ordered by someone else before, and who? No, such information will not be disclosed.
- I have received the seeds with a report sheet. Can I get the sequences on the report sheet electronically? Yes, this data can be obtained from the get confirmation sequence page. You only need to enter the sequence identifier in the first line of the FASTA format sequence.
- The new MTA (pv20050624) with a line fee of 100,- EUR is available on your web site since 24.06.2005. What happens with line orders which were placed before that date? Unpaid invoices need to be paid according to the amount mentioned on the invoice. All invoices issued after 24.06.2005, including those for lines that were in the confirmation pipeline in June 2005, will be based on the fee of 100,- EUR per line.
- I am interested in getting GABI-Kat lines, but how do I know if and when these lines will be available at NASC? Please check the NASC website. Transfer of confirmed GABI-Kat lines to NASC has been initiated in June 2005. The number of lines at NASC will increase over time and you can find numbers on the GABI-Kat News & History page as well as on the SimpleSearch query definition page. Also, for individual lines this information is displayed in the "line availability" field on the "show sequence" page. You can look at an example by searching for insertions in the gene At2g31180; the result is line 067H03 that has been confirmed to contain an insertion in At2g31180 and is only available from NASC.
- If I order a line from GABI-Kat and confirmation is successful, when is that line given to NASC? Usually we deliver T2 seeds to those who order lines at GABI-Kat. It takes another generation to harvest T3 seeds which are finally given to NASC. We need time to process and prepare the T3 seed bags for delivery to NASC, and NASC needs some time to integrate our lines into their seed store and database. As a result, you will get the line significantly before it becomes available from NASC.
- What happens to the executed MTAs that are sent to GABI-Kat? We start the confirmation process after we obtain MTAs which fit an order that has been placed via our web order form. After that, there are various options:
(i) if there is no web order fitting a MTA that reaches us, the MTA may sit forever and nothing happens;
(ii) if your MTA fits a web order, you will receive an e-mail message that the confirmation process has been started, and if the confirmation process is successful you will get back the MTA together with seed from the line(s) you ordered. To reduce administrative workload, we DO NOT send back the MTAs separately;
(iii) in case that ALL lines on a given MTA fail to confirm, no seed is sent to you and therefore the executed MTA stays with us. These MTAs are send back every three to four months.
- I ordered lines from GABI-Kat before, and now I would like to order a few new lines. Do I need to send a new MTA for a new order? Yes, for sure you need a new MTA for other (new, additional) lines! The new line IDs (and the AGI codes or BAC IDs) need to be written on the MTA. You also need to send the corresponding purchase order and you need to submit the new data via our web order form.
- Do you accept payment by credit card? Yes, since September 2009 (and after years of asking for this option) we can also accept payment by credit card! You can use VISA or MASTERCARD. The procedure is as follows:
- What happens if my institution does not pay the line fee? If we can not observe that the line fee for (a) delivered line(s) is paid after a few months, a billing reminder e-mail is sent to the person who received the seed. If there is still no money coming in after that first reminder, a second billing reminder is sent. If there is still no response to the second reminder, the institution of the person who did not pay is "black listed". That means that we will not deliver seed to that institution any more until the case is solved. This consequence is mentioned in the second billing reminder e-mail. The third level is a "snail mail" letter to the administration head of the institution.
- What does the name GABI-Kat stand for? Please see this page for the answer.
- How is the term "T1" interpreted by GABI-Kat?We now use the following nomenclature for seeds and plants, starting with plants being infiltrated. The clarification is consistent with the paper by Ken Feldmann "T-DNA insertion mutagenesis in Arabidopsis: Seed infection/transformation" (Methods in Arabidopsis research, ed. Koncz, Chua & Schell, 275 - 289; World Scientific Publishing 1992).
- T0 plants - the plant which is infiltrated with the agrobacteria (we mean T-zero or T-null!). These plants are setting T1 seed.
- T1 seed - seed produced by T0 plants. These seed are in their testa genetically identical to T0 plants, but contain T1 embryos.
- T1 plants - plants growing out of T1 seed. These plants are setting T2 seed. T1 plants are usually hemizygous for the insertion. T1 plants containing an (intact) insertion are SUL-resistant, and we are isolating DNA from leaves of resistant T1 plants for FST generation.
- T2 seed - seed produced by T1 plants. These seed are in their testa genetically identical to T1 plants, but contain T2 embryos. This is the seed which is sent out.
- T2 plants - plants growing out of T2 seed. These plants are setting T3 seed. T2 plants from a family segregate for the insertion(s) and for resistance. We grow some T2 plantlets to isolate T2 genomic DNA for the confirmation PCR. For lines containing one insertion locus, 33% (statistically) of the resistant T2 plantlets should be homozygous for the insertion (... you do recall Gregor Mendel' law?).
- T3 seed - seed produced by T2 plants. These seed are in their testa genetically identical to T2 plants, but contain T3 embryos .......... In cases in which we have no T2 seed left after confirmation, we deliver a set of T3 seeds. These T3 seeds are those that are sent to NASC. A given T3 set contains seeds of all T2 plants that did produce seed (separatly for each T2 plant).
- How to get a probe to check for the insertion on a DNA gel-blot? We suggest that you amplify a probe from genomic DNA of the lines. We prefer PCR amplification over the preparation of plasmid DNA from large low-copy vectors.
- What is the difference between a single line and a T3-set at NASC? GABI-Kat lines that have been confirmed are transferred to NASC as sets of T3 seeds. Each line within a set is a sister line of the other lines in that set. The T3 seeds are harvested from individual T2 plants from a segregating family. In addition, all T2 plants were (usually) sul-resitant. As a result, the T3 set has a high probability to contain seeds from at least one homozygous T2 plant for a given insertion line.
- What if my question is not answered in the FAQ? E-mail us at infogabi-kat.de. Make sure to use an informative subject header line. Mails that have no subject or include a subject text that looks like SPAM will not be read. In fact, such emails are deleted automatically.
- I wrote e-mail to GABI-Kat and I always got reply e-mails with something like GKT:XXXXXXXXXX or CBT:XXXXXXXXXX in the subject line, what's that? Since August 2003, we used an email management system (OTRS, http://otrs.org/) to keep track of all the email to and from GABI-Kat. The GKT:XXXXXXXXXX or CBT:XXXXXXXXXX is the identifier of the thread, please do not modify it when you reply or forward.
UPDATE: the OTRS system was discontinued in August 2008 and replaced by a simple shared IMAP mailbox. The GABI-Kat Team takes care about answering incoming questions.
- How should I cite GABI-Kat in my publication? Please see this page for the answer.
- How to identify a second insertion in a GABI line? First of all, there are many lines with two or even more insertions. Some are detected by FSTs, others not. There are also lines with more than one confirmed insertion, e.g. 011F01 or 044H05. If you do a phenotypic characterisation, and you detect a phenotype that is NOT segregating with the insertion you know, there is a chance to find a 2nd T-DNA insertion. Please see the Methods page Identify 2nd insertion for details.
- Why do I get seeds which I ordered a very long time ago? While working on the continuous improvement of the data quality at GABI-Kat, we occasionally find seed requests in which users did not get the allele (line) because the insertion was not confirmed initially. If we find logistic errors in our population, we do correct them. As a result, it can happen that you receive seeds for an order placed years ago (we do not ask for the line fee in these cases). Such lines are also donated to NASC, but we want to inform the user who initially wanted the line that "his" or "her" allele has finally been confirmed.
- Are ALL GABI-Kat lines sul resistant? There are very few lines that do contain a T-DNA but are seemingly not resistant. An example is 840D06. In the cases we have observed so far, the respective line is sensitive from the beginning. We assume that the sul marker got damaged during integration. Since many lines do contain several T-DNA insertions, it is theoretically possible that the T-DNA in a given allele does not confer resistance, and the other "normal and intact" T-DNAs have been segregated away. It is also possible that the sul resistance gets silenced like kan in SALK lines. However, in our hands we have not observed such a case so far.