FAQs

 

How much tissue sample do you need for mapping and genotyping?

For general genotyping we require one ear punch or equivalent size piece of tissue per well of a 96 well polymerase change reaction (PCR) plate.  The plate should be clearly labelled on the edge (not over the lid) with a plate/request ID and name. A hardcopy template should be provided with the plate and an electronic copy submitted with the request. Samples should be collected on ice and stored at -20ºC or -80ºC.

For mapping scans we require three ear punches or 0.5cm tail in 1.5ml microfuge tubes or 2ml screw cap tubes. These should be barcoded where possible or clearly labelled with strain, generation and mouse number (the information on the tube should match your request sheet). Samples should be collected on ice or liquid nitrogen and stored at -80ºC.

For Sanger sequencing we require at least three ear punches or 0.5cm tail (please provide more if sequencing > 50 genes) in 1.5ml microfuge tubes or 2ml screw cap tubes. These should be collected, labelled and stored as above.

 

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How long do you keep samples?

We keep genotyping plates for three months. Upon request we keep all mapping samples until completion of a project. These samples will then either be disposed of or shipped back to the client.

 

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In what type of tubes should samples be submitted?

Genotyping samples should be submitted in a 96-well PCR plate that is clearly labelled with your request number or Musterer database plate number on the edge of the plate (not over the lids).

Mapping and whole exome sequencing samples should be submitted in 1.5ml microfuge tubes or 2ml screw cap tubes that are either barcoded or labelled with strain name, generation and mouse number clearly on the side of the tube.

 

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Where are samples dropped off?

We are situated in the Hugh Ennor building, 117 Garran Road in the Australian National University Acton campus. Please advise the front desk of your arrival and we will meet you to collect your samples. If you have access to our area then please place samples in our -20ºC freezer and write your details on the freezer list. Please make sure you have submitted a request form on our task request system prior to submitting your samples.

 

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How much tissue/DNA is required per service/assay?

Please note that these are the minimal amounts specified in the respective protocols. We recommend you provide us with excess when possible:

Service

Amount RNA/DNA required (ng)

Amount RNA/DNA required (ul)

General genotyping

500ng

5µl (100ng/µl)

Amplifluor genome scan

8µg

80µl (100ng/µl)

Fine mapping (average 10 markers)

1µg

10µl (100ng/µl)

Sanger sequencing

50ng/exon

1µl/exon (50ng/µl)

Whole exome next generation sequencing 5ug 35ul (>150ng/ul)
 

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What are the expected timeframes for completion of requests?

Service request

Timeframe *

Genotyping

1-2 weeks

Amplifluor genome scan

2-3 weeks

New assay

2-3 weeks

DNA preparation (plate)

2-3 days

DNA preparation (tube – purified)

1 week

Sanger sequencing

4-6 weeks

Whole exome next generation sequencing

8-12 weeks

*While every endeavour is made to process samples as quickly as possible turnaround times may vary depending on workload.

 

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Can researchers prepare their own DNA for mapping and genotyping?

We do accept DNA prepared outside the facility, although we prefer to prepared it ourselved. If researchers do prepare their own DNA our protocol must be used where possible. We also accept DNA prepared by other methods, but please specify which method you have used.

All nucleic acid extractions conducted outside the facility will be assessed for concentration on the nanodrop and by gel electrophoresis. Investigators are responsible for the cost of this quality control (QC) measure unless they can provide nanodrop and gel or bioanalyzer files at the time of sample submission.

DNA should be stored in 10mM TrisHCl (Tris EDTA (TE) should not be used as a storage buffer).

 

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Can researchers provide their own DNA for whole exome next generation sequencing (NGS)?

Researchers can provide their own DNA however it has to pass our quality control checks of high molecular weight and purity. We require pure, high molecular weight DNA in TE at a concentration of no less than 150ng/ul (preferably higher if possible). We use 3.5ug per sample in the library prep and capture but will need to run some quality tests and validations after sequencing so we ask for a minimum of 5ug/sample. If you have plenty of DNA please provide double this in case we need to rerun your sample. We recommend following the attached protocol or sending tissues on dry ice.

We find that whole ear is providing the best DNA and are using that in our pipeline here. If you do not want to sacrifice the mice then four ear punches are required. We find that unless the tails are really fresh that we get some degradation of the DNA so prefer not to use them.

 

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What quality control (QC) checks do you use for mapping and genotyping?

We run two to four controls with every PCR to ensure only the required fragments have been amplified. We also routinely rerun a percentage of our samples to ensure reproducibility of our assays.

DNA preps are analysed on a nanodrop 1000 to obtain 260/280 and 260/230 readings. For Sanger sequencing the integrity of the DNA is also checked by agarose gel electrophoresis.

 

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Can you design a new genotyping assay?

We have qualified molecular biologists available to design new assays upon request. Reserachers will need to provide complete details of the strain they want to genotype. We will test and optimise the new assay. Please provide the relevant controls for the optimisation and testing.

 

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In what format is genotyping and mapping data provided?

If mice are housed with us and are registered in the in-house database (Musterer) then all genotyping results will be entered directly into the database for easy access.

For ANU clients not using Musterer, results will be attached in excel format to requests lodged in our system upon completion. For all other clientele results will be emailed in excel format. The data pictures will be available upon request.

 

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How many markers do you run for a  genome scan linkage analysis?

This depends on the breeding strategy used to produce the mapping cross. We have several amplifluor single nucleotide variant (SNV) marker sets that are polymorphic between a wide range of mapping crosses. For a low density amplifluor genome scan we will run ~100 to ~160 depending on what strains are being used in the mapping cross.

 

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How many mice are needed for the genome scan?

This depends on the breeding strategy and trait. Generally if the phenotype is recessive then we recommend running ≥8 affected mice and for a dominant trait it is recommended to run 15-20 informative mice.

 

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Can samples be pooled for the genome wide scan?

For the amplifluor genome scan, if the mutant phenotype is very clear then we recommend that running the mice as a pool of affecteds and a pool of unaffecteds where appropriate.

 

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What type of services does the DNA sequencing and bioinformatics pipeline provide?

We offer a mouse SNV detection pipeline. This includes DNA preparation from mouse tissues, library preparation, exome enrichment, paired-end sequencing and bioinformatic analysis.

 

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How are the results from whole exome NGS presented?

Data will be presented as an excel spreadsheet documenting all SNVs found in your sequence data. This will be sorted so that the most likely N-ethyl-N-nitrosourea (ENU) mutations will be at the top of the list. Our bioinformatic pipeline is continuously developing and we will soon be able to provide a full report on sequence quality, median coverage and a list of exons that are not covered in samples.

 

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What QC steps do you take prior to whole exome NGS?

All genomic DNA samples submitted to or extracted by the core facility for library construction will go through quality control (nanodrop spectrophotometer reading and evaluate quality on a one percent agarose gel) prior to library construction.

Post-library QC includes the following services:

  • nanodrop reading
  • q-pcr quantification of library concentration
  • evaluation of library on an agilent bioanalyzer DNA 1000 chip.

These services are included in the total price.

We send the enriched libraries for sequencing to one of our sequencing providers who all follow manufacturer's requirements for running the samples, along with QC at the required steps.

 

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How much of the mouse exome will be covered to sufficient depth for SNV discovery?

Currently we are covering >90 percent of the consensus coding sequences (CCDS) exome at five times coverage or greater with a median read depth of 50 times from data from one lane of an Illumina GAIIx using paired end 75bp reads. We are averaging 40 million reads per lane/sample.

 

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How many samples should be run for single nucleotide polymorphisms (SNP) detection via whole exome NGS?

We recommend running two samples per strain for exome sequencing if the mutation has not been mapped. This greatly increases the chances of locating the causal mutation amongst other incidental mutations and any false positive calls. If the strain has been mapped one sample will be adequate.

 

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Is the sequence of the background inbred strain required prior to whole exome NGS?

We have SNV data for C57BL/6j, C57BL/6s, CBA/Caj, Balb/b and C57BL/10SnJ strains based on exome sequencing of these strains. We also have accumulated SNV data for C3H/CeJ strain based on sequence from successive samples on a mapping cross.

If the required background strain/s is not on one of these backgrounds or on one of the 17 mouse strains sequenced by Sanger (PMID: 19825173) then the sequence of the background strain/s exome will be required..

Updated:  26 September 2017/Responsible Officer:  Director/Page Contact:  Site manager