AGRF Client Portal
I can’t log into my AGRF Account.
You can reset your password via our LIMS sample submission page. Please wait 30 minutes before trying again with the new password. Ensure that:
for data collection, you are trying to login to agrf-data.agrf.org.au
You are located Australia/NZ - if not, we just need to open the site up to you - please contact Customer Care,
You have the correct username – this should match what is in the email sent to you by AGRF
I can’t find my data on the AGRF portal - how can I access my data?
Ensure that you’re at agrf-data.agrf.org.au (not the ftp site) and that you have the username that matches the username in the email sent to you by AGRF. If you can see the data via an sftp server such as FileZilla, but not via the OwnCloud portal, kindly ask one of the bioinformatics team to reset permissions on your client account.
Why am I getting an error when submitting my samples?
When submitting your samples, remember that certain characters are illegal! You can’t use: ? ( ) [ ] / \ = + < > : ; " ' , * ^ | & . Our submission process only allows alphanumeric characters and underscores.
Can I check the progress of my sequencing?
Check your Batch Summary reports (PDF files) in your Data Folder for information on how your sequencing is performing.
AGRF Services
Does AGRF offer a Metagenomics Service?
Yes, we offer a range of Metagenomics and Microbiome services. You can read more about these services on our Microbiome page.
Can we drop off the NGS samples locally?
We have a national sequencing network with laboratories in five states and sample collection points located across Australia. To find out where your nearest sample collection point is click here.
Does AGRF provide single cell RNA Sequencing service in Brisbane?
Our 10x Genomics Chromium is located in Melbourne, but we can work with you on preparing the samples locally, for sequencing on our Illumina NovaSeq platforms.
What is the minimum amount of sample that you can accept?
Sample requirements differ for each service. You can find information on minimum sample amounts for your specific needs on our Service Guide page.
Can you return/store the samples after processing?
Samples are stored with AGRF for 3 months after you receive your data. If you wish for your samples to be returned, you must let us know when you are submitting your samples. At the completion of processing, we will return your samples by post, if requested, using an Australia Post Express Post satchel at ambient temperature. If required, we can return samples using Dry Ice, but this will incur a $150 charge. If we do not hear back from you by the date specified in your data delivery email, we will discard your samples.
Can you help prepare custom reports?
Yes, our Bioinformatics Team can help you prepare custom reports to suit your individual goals. For more information, please check out our Bioinformatics page.
16S Service
What kind of samples can be accepted for the microbial ID 16S service (Sanger Sequencing) and how must they be submitted (e.g. can samples be submitted as a culture)?
The 16S Sanger service is for bacterial samples only, fungal samples cannot be processed through this service. Samples must be submitted in an aliquot of PrepMan buffer (bacterial colony suspended into the buffer) or as extracted DNA. Bacterial cultures cannot be accepted.
Bioinformatics
Where can I find the bioinformatic analysis results I requested?
All bioinformatic results are transferred to agrf-data-agrf.org.au, there is a results folder where you can find all the bioinformatic results.
What tools can be used for my analysis?
The team can work with online tools, desktop applications from Windows or Mac or packages requiring an HPC infrastructure.
How do I open a gff3 file in Excel?
A General Feature Format (GFF) file is a simple tab-delimited text file for describing genomic features. You can view it in any text viewer but you need proper tools (such as IGV) to view the features in the reference sequence.
RNA-Seq: Why do all of my adjusted p-values say ‘1’?
Since we are testing the significance of many genes in the one experiment, we must first correct for multiple hypothesis testing, this means that p-values that may have been considered significant if tested on their own are now no longer. Read about multiple hypothesis testing online here.
RNA-Seq: Why are no differentially expressed genes found in my dataset?
Another reason that p-values can reach close to 1 or 1 is due to the shrinkage restrictions made by the quasi-likelihood F-test which is a common method implemented in edgeR for modelling RNA-Seq data. Essentially, if there are many genes in the datasets that show a high level of stochasticity across all samples, then the likelihood of a gene that happens to have a tight clustering of samples split by group, is said more to be by chance rather than actual biological variation between the groups. Datasets with high levels of variation over many genes may not show any genes of differential expression.
RNA-Seq: My dataset clusters into two distinct groups but these aren’t the groups I supplied.
This could be due to using different sexes in the sample set. We may be able to incorporate this as a batch effect if there are enough of both sexes in each group. One way to check if this is the source of the clustering is to look at the XIST gene (which is expressed highly in females and lowly in males).
Exome & WGS: How is coverage calculated?
We provide mean and median coverage values. These are derived as a mean-of-mean and mean-of-median over each interval over a design file. Therefore, if a design file has five exons, and we have a sample alignment information with mean coverages of 30, 100, 10, 120, and 40 then our mean value will be 80x and our median value will be 40x. A two-fold difference between the mean and median would be of a concern and would suggest a low library size. We should expect the mean and median values to be within 1.5x of each other.
Exome & WGS: What does (no PCR) mean in my report?
We also calculate coverages after removing duplicate reads. Duplicate reads are determined by two reads from the same sequencing library having the exact same alignment.
Exome & WGS: How do I verify what reference my dataset was run on?
This should be part of the ‘references’ tab in the pdf report provided. You can verify the value by opening a vcf file and searching for ‘contig’ in the header (the lines that start with “##”). Check for the assembly tag on this line.
Microbial Profiling: What primers are used for amplification?
The information will be provided in a report along with the data delivery notification email.
Microbial Profiling: Why didn’t results show both fungi and bacteria in a sample?
The AGRF primers are specific to a domain. Therefore, you wouldn’t notice the other, though they are present in the samples. Example: if you use 16S Microbial Profiling service you will notice only bacteria not Fungi. Whereas, if you use the ITS service you get only Fungi not bacteria. If you need a full profile from different domains, you can use our Metagenomics Service.
Microbial Profiling: Why are there unclassified reads?
The unclassified reads may be host amplifications, or not have annotations in the database. To address this, we provide NCBI results as supplementary in a file named ‘mg_blast.xlsx’.
Genotyping by Sequencing (GBS)
Why perform an establishment phase?
The establishment phase determines a suitable library preparation method – specifically the optimal enzyme combination and size selection area. AGRF has a suite of eight enzyme combinations commonly used in GBS library preparation.
The standard establishment will assess all eight enzyme combinations to determine the best combination based on absence of repeat regions within the size selection area as well as amplification success.
The premium establishment will assess all eight enzyme combinations to determine the best combination, a library is then prepared using this enzyme combination and assessed over two size selections (narrow and wide) to determine the most informative size selection.
Presence of repeat regions can reduce the amount of useable sequence that can be interrogated for SNP commonality.
What sequence depth is required for GBS?
GBS does not require a specific coverage or depth of sequencing, however it does require high quality sequence. Coverage and sequencing depth are influenced by size and complexity of the genome, enzyme selection and size selection – another reason why the premium establishment can be so valuable.
We favour the NextSeq500 for sequencing GBS libraries for following reasons:
The 150bp read length is perfect for our narrow and wide size selections – our final library size consists of fragments which are 117bp (narrow size selection) or 150bp (wide size selection).
The flowcell configuration means there is no requirement to batch projects together, each project is its own run meaning no delays.
Can GBS be customised? I want to replicate the methods in a publication?
The AGRF GBS service can accommodate some custom requests from clients – where a client requests a variation on enzyme combinations, size selections or sequencing platform please forward these to the Genotyping team for review. If attempting to replicate a publication, please also provide this so that we can assess if this can be achieved.
What are the sample requirements for GBS?
The success of GBS is contingent on the quality of the input DNA.
DNA should be a high molecular weight (>20 kb), free of RNA and not form streaks when assessed by gel electrophoresis.
DNA should be eluted in RNase/DNase free water.
DNA which is viscous or contains proteins will be less accessible to enzymes reducing their contribution to the final library.
Using degraded DNA can cause false polymorphism detection in the sequence analysis, as the analysis is batch based this can impact all samples.
The quantity required varies between the standard establishment, premium establishment and batch processing phases – please consult the service guide.
Genotyping Fragment Analysis and Separation
What size standards are available?
AGRF utilises the LIZ500 and LIZ1200 size standards. The LIZ600 size standard is available upon request. For the submission of Promega kit products, the customer must supply the size standard, and allelic ladder.
What instrument is used?
Capillary electrophoresis is performed on the Applied Biosystems 3730, with a 50cm array and POP-7 polymer.
What fluorescent labels can I use?
AGRF recommends markers are labelled with the fluorescent dyes FAM, VIC, PET and NED (with LIZ or ROX reserved for the size standard). Fluorescent primer pairs labelled with VIC, PET and NED dyes are proprietary to Applied Biosystems. AGRF can order these primers from provided sequence. Please contact us if you wish AGRF to order primers. Alternative labelled primers from alternate suppliers compatible with the DS33/G5 matrix can be utilised. Please contact us if you have any questions regarding your fluorescent dyes.
Human Cell Line ID
Can non-human cell lines use this service?
No – This service is only suited to human samples and will not indicate presence of DNA from non-human cell contamination.
What percentage match is considered a cell line ID match?
Cell lines with greater than an 80% match are considered related; derived from a common ancestry. Cell lines with between 55% to 80% match are most likely unrelated and may require further profiling for authentication of relatedness. If you require a higher resolution assay option, please contact AGRF to discuss options. The International Cell Line Authentication Committee (ICLAC) recommends including the use of at least eight core STR loci and application of match criteria (80% match threshold) to allow for a small amount of genetic drift in some cell lines.
Illumina SNP and Methylation Service
What format do you require the samples to be shipped in?
For less than 96 samples, tubes can be submitted. It is recommended for >96 samples, samples are sent in a 96-well plate. Please ensure the samples are in column format (i.e. A1-H1, A2-H2 etc) and the plate is heat sealed or appropriately sealed. We recommend plates are sent on dry ice, to prevent leakage
For samples that fall below 50ng/µl do we submit much higher volumes or should we concentrate the DNA instead? Alternatively, are you able to concentrate the DNA for us?
Although we do request samples be submitted at ≥ 50ng/µl, we can accommodate samples below the requested concentration. As long as you submit a total quantity of 1-1.5µg, we can concentrate this into our required volume as part of the normalisation process.
Are we able to run theIillumina MethylationEPIC array on FFPE samples? What is the minimum input?
The MethylationEPIC array is suited to FFPE samples. For these samples, an Illumina restoration is required. AGRF does not offer the Illumina restoration as a service. For FFPE samples undergoing restoration, you will also need to perform the bisulfite conversion in your own lab. Please note, for samples submitted as bisulfite converted DNA we are unable to perform a DNA QC assessment. The Illumina process requires 4µl of the converted product. We require submission of a minimum of 5µl of converted product for the illumina MethylationEPIC array.
Can WGA samples be used for Illumina SNP and Methylation service?
We strongly discourage customers from submitting whole genome amplified (WGA) samples. WGA samples have an unpredictable success rate. However, if you must submit WGA samples, the following observations may be helpful:
Unlike gDNA samples, the final concentration of WGA samples does not correlate to sample quality. After WGA, it is possible to have high DNA concentration that is not representative of the original DNA sample — particularly if the starting concentration was low or if contaminating DNA was present.
The best metric correlating WGA sample quality is the final quality/quantity of the sample before amplification. It is recommend using a minimum of 10 ng of gDNA for the WGA reaction. (Better results have been observed with ≥ 50 ng of gDNA).
If a combination of WGA and non-WGA samples are submitted as a single batch, it is recommended the sample sets be separated for analysis, due to potential differences in the clustering of the SNPs for allele calling.
Microsatellite Genotyping
What if there are no microsatellites or limited number for my species of interest?
AGRF can assist with microsatellite discovery by using the Illumina MiSeq. A genome can be scanned at reasonably low coverage and still provide microsatellite sequence information allowing for marker design. For more information on this service, please contact us.
Sample Preparation
My samples are frozen, should I thaw them?
No. If your un-extracted sample is currently frozen, it’s best to keep it frozen until the point of DNA extraction. Thawing/refreezing samples, or leaving samples at room temperature prior to extraction will result in degradation of the DNA, and may lead to lower-quality results.
Should I treat my RNA samples before sending for Library Prep?
Before submitting RNA samples for RNA library preparation and sequencing, we recommend that you do a DNase treatment.
How should I label my tubes?
If you are hand writing your tube labels, here are a couple of tips:
Labels written with a red marker are very difficult to read.
Try to use a thin black marker pen to make labelling easier to read.
Use a unique identification name on the SIDES, but keep the TOP label simple and easy to read.
To keep it simple, use your initials followed by submission number and sample number eg. JJ13-1 onwards.
How should I quantify the concentration of nucleic acids in my sample?
Need to accurately quantify your samples? Use fluorometric QC for more consistent results.
How can I remove dead cells before Single Cell Analysis?
You can remove DEAD CELLS in a single cell suspension using MACS Dead Cell Removal Kit. (We get ours from Miltenyi Biotec)
Do you have any tips for dealing with high molecular weight DNA?
High molecular weight DNA should be handled very carefully to avoid shearing.
It should not be vortexed.
Use wide-bore tips for mixing (you can just cut the end off a regular tip to make it wider).
Ensure slow pipetting (3-second strokes in wide-bore tips).
Sanger Sequencing
What buffer should I use for my Sanger samples?
Elution buffers may inhibit Sanger sequencing. It’s usually best to elute your DNA sample in water.
Will residual ethanol affect my Sanger results?
Residual ethanol will affect the quality of your Sanger sequencing. Be sure to dry out your samples completely after any ethanol-containing wash or precipitation step. This ensures that no ethanol remains in your DNA sample.
Is it possible to request PD re-runs, and what is the time frame and cost involved?
PD re-runs can be requested within 2 weeks of the original samples data being sent. Re-runs will be free of charge, however, large batches of re-runs will not be accepted until a smaller portion are re-run (2-3 samples) and only if those produce better results. If the results have not improved from the re-run, the remaining batch for a large re-run will not be accepted.
Shipping Samples
How should I ship my 96-well plates to AGRF?
We recommend shipping on dry ice to minimize the chance of leakage during transit due to air pressure changes on airplanes.
