Next Generation Sequencing Resources

AGRF Next Generation Sequencing services are offered using Illumina HiSeq and MiSeq platforms.  

Our services are run by experienced staff who are easily contactable via phone or email. We are available to give advice at any stage of your project and can assist you in finding a suitable sequencing solution.

Our resources are split into the following sections for easy navigation:

Submitting samples to AGRF

Understanding our services

Submitting samples to AGRF

The following documents outline the process for submitting your samples to our next generation services. Below you will find our guides for sample submission requirements and navigating our online submission system. 

Understanding our services

Next Generation Sequencing Applications

 

DNA Sequencing Applications

RRBS

Reduced Representation Bisulfite Sequencing (RRBS) is a comprehensive analysis method and an economical alternative to whole genome bisulfite sequencing-requiring approximately 50 fold less sequencing.

It provides strand specific single-base resolution of DNA methylation (5 methylC) across a broader genomic span than predesigned arrays.

The method employs next generation sequencing with a library preparation method using restriction digestion of genomic DNA with the methylation insensitive restriction enzyme MspI. This has a recognition site of “CCGG” to enrich the sample for genomic fragments with a high frequency of potential CpG methylation sites, thereby effectively capturing ‘CpG islands’.

 The method is flexible in design being suitable for both model and non-model species with a reference genome, and delivers a high-throughput method well suited to genome-wide DNA methylation studies.

Details:

NuGEN Ovation RRBS Methyl Seq kit
Suitable for genomes with a reference (human, mouse, rat and other mammals).
Not recommended for plants due to their methylation mechanisms.

Sample submission details:

•         1 μg of DNA per sample requested (≥20 ng/μl)

We recommend

  • 100 bp single read sequencing

Reads

  • 30M reads (or read pairs) per sample.

Given the nature of the method, variations to this recommendation are possible ot meet your experimental needs. We’ll be happy to discuss what best suits your aims.

Bioinformatic analysis options are available upon request.

For more information, please download the service info sheet.

 Shotgun

 Suitable for paired end and single end sequencing for genomes and long range PCR products (>5kb). Pipin Prep size selection is available.

Our service includes Shotgun library preparation and QC.

Nextera
Available for high throughput, low input DNA sample preparation. This library type is suitable for small genomes and amplicons (300bp). Using a Nextera library allows for up to 96x sample indexing.

 

Exome enrichment
Using exome enrichment you are able to perform targeted re-sequencing of coding regions for human or mouse genomes. This is useful for screening the high value content of genomes, economically.

Exome sequencing at AGRF includes sample quality control, library preparation, exome capture and sequencing on the HiSeq 2500 with 2x100 bp paired-end reads.

We can also provide options for 50X and 100X mean coverage levels and low DNA input Agilent SureSelect Exomes are guaranteed to meet minimum specifications of the Agilent Certified Service Provider program.

 

Custom Capture
Our custom capture libraries allow you to design custom captures to sequence specific genomic regions of interest. This can be beneficial for projects when you have specific targets in mind.

 

Mate-pair
This library preparation is used for applications such as de novo sequencing and structural variant detection. The library prepares long insert libraries from 2-10kb and is compatible with paired end sequencing.

Our service includes mate pair DNA library preparation and QC. We also offer a range of size selection options of 2-4kb, 5-7kb and 8-10kb mate-pair span in gel free and gel size-selected options.

 

ChIPSeq
This is an optimised library preparation for when only small amounts of DNA are available, such as following a Chromatin immunoprecipitation. This library is suitable for applications down to 10ng.

This library can be used for analysing the protein interactions with the genome, like transcription factors.

Our service incudes ChIPSeq DNA library preparation and QC.

 

 

RNA Sequencing Applications

 

mRNA or stranded mRNA
mRNA or stranded mRNA is the preparation of mRNA sequence libraries from total RNA via purification of PolyA RNA. Options are available for strand specific libraries for improved genomic information. This library preparation would be used for application such as transcriptome analysis or gene expression quantification.

 

Whole Transcriptome
This is the preparation of a whole transcriptome library for analysis of the coding and non-coding RNA. The process incorporates ribosomal RNA depletion. This service is currently for human, mouse and rat only.

Small RNA
This library is the incorporation of small RNA molecules (19-35 nucleotides). This enables discovery and expression profiling of microRNAs and other small non-coding RNA in any organism.

 

Library QC
We also offer the option to qualify a library you have prepared yourself prior to sequencing with our qPCR. This allows you to create your own libraries and outsource to us for the sequencing component of your research.

 

RNA Seq FAQ

 

Diversity Profiling
Our Diversity Profiling service is a method of identifying the relative proportion of organisms present in a mixed community.

To do this, AGRF will take your sample (either raw sample, or extracted gDNA) and PCR amplify a region of interest from your mixed community. We will then pool and sequence the barcoded amplicon(s) on the MiSeq platform utilising Illumina’s Paired End Chemistry.

Access the DivPro Sample Preparation Guide here.

DivPro FAQ


Whole Genome Sequencing
The whole genome sequencing service is offered on a per sample basis at 30x coverage. 

Genomic DNA samples are submitted to your local AGRF node, for library preparation and Illumina HiSeq X Ten sequencing by KCCG.

Service Specifications
(per genome)
 
Sample required

TruSeq Nano library

100uL of 20ng/uL or greater purified genomic DNA

TruSeq PCR-free library

100uL of 30ng/uL or greater purified genomic DNA
Minimum sample number Starting from 1x genome
Coverage

Human: 30x mapped equivalent. Also available in 60x and 90x

(i.e.100GB units)

Non-human: coverage dependent on organism genome size.

Sequencing yield >100GB, 2x 150bp paired end reads
Data format FASTQ, delivered by SFTP download
Analysis Data will be QC checked only, no added analysis included

Why whole genome sequencing?

Consistent coverage
Coverage across the exome is highly variable: although WES is typically undertaken at high mean coverage (>100X), >20X coverage achieved over only ~85% of targeted coding regions. WGS provides very consistent coverage - at 40X mean coverage, >96% is covered at >20X depth.
 
Copy number variation (CNV)
The consistent and genome-wide coverage provided by WGS makes it straightforward to call copy number variants. Variable coverage – including gaps over noncoding regions – makes CNV detection challenging with WES.
 
Structural variation
WES can seldom define structural variation. Chromosomal modifications, such as translocations and inversions, can be precisely defined by WGS, often with single-nucleotide precision.
 
Comprehensive coverage
WGS covers all regulatory regions, noncoding RNAs, and every exon of every protein, regardless of annotation (i.e. the mappable genome). Whole genome data can be re-queried as new functional elements are identified or as answers to new questions are sought.
 
Research value
Whole genomes are agnostic to our currently limited understanding of genome function and are therefore much more valuable for research purposes.
 
Diagnostic yield
Diagnostic yield from clinical whole genomes is significantly higher than exomes, due to their consistent coverage across exons, splice sites, and the detection of noncoding variants and CNVs.
These advantages were directly sourced from the KCCG website

This service is currently available for research purposes only. Researchers are responsible for ensuring that all relevant ethical approvals have been obtained prior to providing samples.

Globin Depletion
We use the Illumina TruSeq Stranded Total RNA with Ribo-Zero Globin to remove globin mRNA and ribosomal RNA from a blood samples. This cuts substantial amounts of RNA from the template to be sequenced enabling deeper sequencing into the mRNA linked to the biological or disease question.

This protocol enriches the remaining biologically informative transcripts in RNASeq libraries, and gives researchers an expression view that’s far clearer on relevant changes, as well as potentially detecting gene regulations not possible in non-globin reduced samples.


How it works

The Ribo-Zero Globin depletion protocol uses biotinylated probes that selectively bind globin mRNA in combination with nuclear and mitochondrial ribosomal RNAs (rRNA). RNA’s bound to the probes are captured by magnetic streptavidin beads and removed.


RNAs targeted for removal are:

Globin mRNA

Hba-a1

Hba-a2

Hbb

Hbg1

Hbg2

Mitochondrial rRNA       

18S

12S

Nuclear rRNA

28S

18S

5.8S

5S

Following depletion of globin mRNA and rRNA, the remaining sample is comprised of regular gene messenger transcripts and long non-coding RNA species.  They are incorporated into a sequencing library by the TruSeq Stranded Total RNA method, omitting the now unnecessary polyA steps.  Sequencing requirements will depend on the application, however AGRF generally recommends a minimum of 30 million reads per sample.

This protocol is compatible with the PAXgene Blood RNA Tube system.  This system is used for collection and storage of blood, stabilizing intracellular RNA.  

News

NovaSeq 6000 Service now available!

15 May 2017

An exciting addition to the AGRF technology portfolio has arrived and is currently undergoing rigorous validation testing.

AGRF’s NovaSeq 6000 sequencing hardware is the first of its kind that is operational in Australia and opens the door to broader, more comprehensive genome studies.

Read more ›