SAMPLE PREPARATION, QUANTIFICATION & QUALITY REQUIREMENTS

 

Illumina MiSeq™ Sequencing APPLICATIONS

Application

Method of Library Preparation

Type of Sample to Supply

Illumina MiSeq Read lengths recommendations

Minimum Amount
Required

Minimum Concentration
Required

Buffer

Genomic DNA Sequencing of small genomes
De novo
assembly

 

Illumina TruSeq™ DNA PCR-Free Library Preparation

 


Illumina Nextera™ Mate Pair Library Preparation (inserts 2-8Kb)*

 

Fungal, bacterial, viral and smaller complex genomes

Illumina MiSeq V2
(2x 250 base PE) 1

Illumina MiSeq V3
(2x 300 base PE)2,3

1.5µg DNA
350 bp insert
2.5µg DNA
550 bp insert


1.0-4.0µg DNA

20ng/µl

 

 

200ng/µl

 

10mM Tris-HCl, pH8.5 or molecular grade water

Genomic DNA Sequencing of small genomes

Re-sequencing

 

Illumina TruSeq™ DNA PCR-Free Library Preparation

 

 

 Illumina TruSeq™ DNA Nano Library Preparation

 

Illumina Nextera™ DNA Library Preparation

Illumina Nextera XT™ DNA Library Preparation

 

Fungal, bacterial, viral and smaller complex genomes

Illumina MiSeq V2
(2x 250 base PE) 1

Illumina MiSeq V3
(2x 300 base PE)2,3

1.5µg DNA
350 bp insert

2.5µg DNA
550 bp insert


150ng DNA 350 bp insert
150ng DNA 350 bp insert

100ng DNA

 
1.5ng DNA

20ng/µl

 

 

20ng/µl

 

 

 10ng/µl

0.2ng/ µl

10mM Tris-HCl, pH8.5 or molecular grade water

Whole Metagenomic Sequencing

llumina TruSeq™ DNA PCR-Free Library Preparation

 

 

 Illumina TruSeq™ DNA Nano Library Preparation

 

 Illumina Nextera™ DNA Library Preparation

 

Microbial environmental samples containing a population of microbes

Illumina MiSeq V2
(2x 250 base PE) 1

 Illumina MiSeq V3
(2x 300 base PE)2,3

1.5µg DNA
350 bp insert

2.5µg DNA
550 bp insert

 
150ng DNA 350 bp insert
150ng DNA 350 bp insert

 100ng DNA

 

20ng/µl

 

 

 

20ng/µl

 

10ng/µl

10mM Tris-HCl, pH8.5 or molecular grade water

PCR Amplicon Sequencing

 

Illumina Nextera™ DNA Library Preparation

 
Illumina Nextera XT™ DNA Library Preparation

 

Large Amplicons ≥ 2Kb

Illumina MiSeq V2
(2x 250 base PE) 1

 

Illumina MiSeq V3
(2x 300 base PE)2,3

100ng DNA

 

 1.5ng DNA

10ng/µl

 

0.2ng/ µl

10mM Tris-HCl, pH8.5 or molecular grade water

Custom Amplicon Sequencing

 

Illumina Two Step PCR Amplicon Approach

using the Illumina Nextera XT ™ DNA Primers for the second PCR step

 Client is responsible for carrying out the first PCR step using custom made Illumina tailed primers (Additional information is provided upon scoping of the project)

Small Amplicons ≤ 450bp

Illumina MiSeq V2
(2x 150 basePE) 1

 

Illumina MiSeq V2
(2x 250 base PE) 1

Run length dependent on the size of the amplicons being sequenced

1ng in 25µL of the amplicon generated from the first PCR step

 

N/A

10mM Tris-HCl, pH8.5 or molecular grade water

Metagenomic 16S Amplicon Sequencing

V4 hypervariable region

16S Amplicon Library Preparation

(Additional information is provided upon scoping of the project)

Genomic DNA or PCR Amplicon of 16S gene
Amplicon size = 253-254 bp

 

 

Illumina MiSeq V2
(2x 150 basePE) 1

 

50ng Genomic DNA or 10ng PCR Amplicon 16S gene

10ng/µl Genomic DNA or 2ng/µl PCR Amplicon 16S gene

10mM Tris-HCl, pH8.5 or molecular grade water

Metagenomic 16S Amplicon Sequencing
V3-V4 hypervariable region

Illumina Two Step PCR Amplicon Approach Using the Illumina Nextera XT ™ DNA Primers for the second PCR step

Client is responsible for carrying out the first PCR step using custom made Illumina tailed primers (Additional information is provided upon scoping of the project)

 

Genomic DNA or PCR Amplicon of 16S gene Amplicon size = 253-254 bp

 

Illumina MiSeq V2
(2x 250 base PE) 1

 

1ng in 25µL of the amplicon generated from the first PCR step

 

N/A

10mM Tris-HCl, pH8.5 or molecular grade water

Small RNA Sequencing

-  Illumina TruSeq™ Small RNA Library Preparation ¥ (Refer to preparation of RNA samples)

 - Illumina TruSeq™ Stranded Total RNA Library Preparation

Total RNA

Illumina MiSeq V2
(2x 25 base PE) 1

 

Illumina MiSeq V2
(2x 150 base PE) 1

1.5µg Total RNA

200ng/µl

10mM Tris-HCl, pH8.5 or molecular grade water

RNA Sequencing
Gene Expression Analysis
Transcriptomics
#

-  Illumina TruSeq™ RNA Library Preparation

 -  Illumina TruSeq™ Stranded Total RNA Library Preparation

 -  Illumina TruSeq™ Stranded mRNA Library Preparation#

Total RNA

Illumina MiSeq V3
(2x 75 base PE)2

 

Illumina MiSeq V2
(2x 150 base PE) 1

 

1.5µg Total RNA

 

1.5 µg Total RNA

 
1.5 µg Total RNA

20ng/µl

 

 200ng/µl

 
200ng/µl

10mM Tris-HCl, pH8.5 or molecular grade water



NOTES:

* Otago Genomics & Bioinformatics Facility (OGBF)  will prepare the Illumina Nextera™ Mate Pair Library Preparations for delivery to MGS for Illumina MiSeq runs. Libraries with inserts >8Kb must be authorised by OGBF before quoting or finalising SA. MGS does not prepare Illumina Nextera™ Mate Pair Libraries.

 #  OGBF will prepare the Illumina TruSeq™ Stranded mRNA Library Preparations for delivery to MGS for Illumina MiSeq runs. MGS does not prepare Illumina TruSeq™ Stranded mRNA Libraries.

¥ Client must purchase the whole Illumina TruSeq small RNA kit which contains reagents to prepare 24 libraries. This is due to the lack of demand for this library preparation method.

1.    Version 2 Illumina MiSeq chemistry. Refer to “Illumina MiSeq Run Options” for details.
2.     Version 3 Illumina MiSeq chemistry. Refer to “Illumina MiSeq Run Options” for details.
3.     This run length has yet to be validated by NZGL services.

Preparation of DNA Samples

It is recommended that clients use column based purification methods to extract the genomic DNA. DNA samples should be treated with RNase prior to purification on the column. Drying the column after the wash step with a second spin is essential to remove excess ethanol, which will interfere with library preparation. Avoid the use of organic extraction methods, such as phenol or Trizol, which can interfere with the enzymatic reactions used during the library preparation protocols.

The client MUST quantitate the Genomic DNA or PCR Amplicon and assess the quality of the DNA prior to the delivery of samples for Illumina MiSeq™ sequencing.

~150ng of genomic DNA samples must be run on a 0.5-1% agarose gel with a high molecular weight ladder. MGS will NOT run a gel when the samples arrive at the service.

PCR Amplicons must be run on either a 1-2% agarose gel with a ladder containing similar band sizes to your products, or bioanalyzer e.g. Agilent 2100 Bioanalyzer, if the client has access to this instrumentation, using either the Agilent DNA 7500 or Agilent DNA 12000 Labchip Kit.

 

Preparation of RNA Samples

Total RNA should be extracted using a column purification method. Organic extraction methods, such as phenol or Trizol, are discouraged for the purification of total RNA. Tissues with a high fat content and certain embryonic tissues require an organic step to remove the excess amount of these other bio-molecules. For this we recommend the use of one of the following Qiagen kits: Qiagen RNeasy Lipid Tissue Mini Kit, Qiagen RNeasy microarray tissue mini kit, or Qiagen miRNeasy mini kit. All three of these kits include an organic step prior to column purification. Residual DNA can also be removed during the purification process when using any of these three kits.

It is recommended that RNA should be provided as total RNA for small RNA sequencing. However you can also supply previously isolated micro RNA as starting material. Supply the entire fraction of small RNA purified from 1-10µg of total RNA.

 The total RNA samples must be run on a bioanalyzer e.g. Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Nano Labchip Kit, if the client has access to this instrumentation. Alternatively run the total RNA on a formaldehyde 1% agarose gel with an RNA 6000 ladder and measure the integrity of the RNA upon staining with ethidium bromide.

 It is very important to use high-quality RNA as the starting material. The use of degraded RNA can result in a low yield, over-representation of the 3’ ends of the RNA molecules, or failure of the library preparation. A DNase step must be included as part of the RNA isolation method. The presence of DNA contamination may result in an underestimation of the amount of RNA used.

 

Client Prepared Libraries

MGS will accept client prepared libraries, however an NZGL disclaimer must be included in the NZGL service agreement, as we are not able to guarantee the quality or quantity of data generated from client prepared libraries.

 Client prepared libraries must be run on a bioanalyzer e.g. Agilent 2100 Bioanalyzer, if the client has access to this instrumentation, using either the Agilent DNA 1000 or Agilent DNA High Sensitivity Labchip Kit. If the client does not have access to a bioanalyzer then MGS will QC check the libraries upon arrival at our facility.

Client Sample & Library QC Checks

Samples and client prepared libraries cannot be received by MGS without pre-submission of QC documentation and sample approval by MGS.

Genomic DNA and Amplicon samples: The client must supply MGS with an electronic copy of the 1-2% agarose gel image, or the summary report from the bioanalyzer, and the fluorometer or spectrophotometer concentration and OD260/280 ratio reading.

Quality Standards

High quality genomic DNA should run as a high molecular weight band on a 1-2% agarose gel, with the majority of DNA greater than 10Kb in size and with minimal lower molecular weight smearing. If the majority of the DNA is below 10Kb or smearing is visible, this suggests that the DNA is degraded or lower molecular smearing can be indicative of the presence of RNA contamination. The MGS will not accept your samples for sequencing if visible smearing is seen.

The spectrophotometer OD260/280 ratio should be 1.8-2.0.

 

RNA samples: The client must supply MGS with an electronic copy of the formaldehyde agarose gel image, or summary report from the bioanalyzer, and the fluorometer or spectrophotometer concentration and OD260/280 ratio reading.

Quality Standards

Bioanalyzer Trace: An RIN value greater than 8 is required, or greater than 7 for plant RNA and rRNA ratio (28S/18S or 23S/16S) of 1.5 to 2.5.

Formaldehyde 1% Agarose: High quality RNA shows a 28S rRNA band at 4.5Kb that should be twice the intensity of the 18S rRNA band at 1.9Kb. Both kb determinations are relative to a RNA 6000 ladder. mRNA will appear as a smear from 0.5-12Kb.

The spectrophotometer OD260/280 ratio should be 1.8-2.2.

 

Client prepared libraries: The client must supply MGS with the summary report from the bioanalyzer.

Quality Standards

High quality libraries should have a clean single peak as follows with no noticeable adapter dimer or PCR primer peaks:

~470-670 bp for libraries prepared using the Illumina TruSeq™ DNA PCR-Free or Illumina TruSeq™ DNA Nano Library Preparation methods.
~250-350 bp for libraries prepared using the Illumina TruSeq™ RNA or Illumina TruSeq™ Stranded Total RNA Library Preparation methods.
~175-700 bp for libraries prepared using the Illumina Nextera™ or Illumina Nextera™ XT Library Preparation methods.
~380-400 bp for libraries prepared for 16S amplicon sequencing- V4 hyper-variable region
~570-600 bp for libraries prepared for 16S amplicon sequencing- V3-V4 hyper-variable region
~140-150 bp for libraries prepared using the Illumina TruSeq™ Small RNA Library Preparation method.

Library Preparation Methods

Below is a list of the library preparation methods used by the Massey Genome Service to prepare libraries for the Illumina MiSeq™ and Illumina HiSeq™ instruments.

Illumina TruSeq™ DNA PCR-Free Library Preparation

This method uses mechanical shearing to randomly fragment the genomic DNA. Magnetic bead size selection will be used to select fragments with an average size of 350bp or 550bp and then barcoded Illumina adapters will be ligated onto each end of the fragments. This library preparation method does not include a PCR enrichment step. 

This method is suitable for preparing libraries from genomic DNA for whole genome sequencing (De novo assembly or re-sequencing) and whole metagenomic sequencing.

 

Illumina TruSeq™ DNA Nano Library Preparation

This method uses mechanical shearing to randomly fragment the genomic DNA. Magnetic bead size selection will be used to select fragments with an average size of 350bp or 550b and then barcoded Illumina adapters will be ligated onto each end of the fragments. The library products are then enriched for products which are fully ligated. 

This method is suitable for preparing libraries from genomic DNA for whole genome sequencing (Re-sequencing) and whole metagenomic sequencing.

 

Illumina Nextera™ DNA Library Preparation

This method use enzymatic shearing to randomly fragment the genomic DNA. Barcoded Illumina adapters will be added onto each end of the fragments during enrichment PCR. 

NOTE: Preparing samples ready for the Illumina Nextera™ DNA Library preparation, please do not send the samples in a buffer which contains EDTA. EDTA is known to be an inhibitor in enzymatic reactions and could inhibit the enzymatic fragmentation used in the Illumina Nextera ™ DNA Library preparation method.

This method is suitable for preparing libraries from genomic DNA for whole genome sequencing (Re-sequencing), whole metagenomic sequencing and PCR amplicon sequencing (amplicon ≥ 2Kb).

 

Illumina Nextera™ XT DNA Library Preparation

This method use enzymatic shearing to randomly fragment the genomic DNA. Barcoded Illumina adapters will be added onto each end of the fragments during enrichment PCR.

NOTE: Preparing samples ready for the Illumina Nextera™ XT DNA Library preparation, please do not send the samples in a buffer which contains EDTA. EDTA is known to be an inhibitor in enzymatic reactions and could inhibit the enzymatic fragmentation used in the Illumina Nextera ™ XT DNA Library preparation method.

This method is suitable for preparing libraries from genomic DNA for whole genome sequencing (Re-sequencing) of small genomes, whole metagenomic sequencing and PCR amplicon sequencing (amplicon ≥ 2Kb).

 

Illumina TruSeq™ RNA

This method generates mRNA targeted libraries from Total RNA, and uses oligo dT beads to extract poly-adenylated mRNA from total RNA. RNA must be of high integrity so as to extract full length transcripts, and minimise 3’ bias. Does not provide strand information on RNA transcripts.

This method is suitable for preparing libraries from total RNA for RNA sequencing, gene expression analysis and transcriptomics.

Illumina TruSeq™ Stranded Total RNA

This method captures both coding RNA, as well as multiple forms of non-coding RNA. Strand information on RNA transcripts is obtained. This method is suitable for a range of samples including low-quality and FFPE. This method employs Ribo-Zero ribosomal reduction chemistry to eliminate the ribosomal fractions. This method works best on human, rat and mouse samples, but can be used on other samples but is less optimal.

Please see the Ribozero-gold species compatibility table on the epicentre website:

http://www.illumina.com/products/rrna-removal-kit-species-compatibility.html

This method is suitable for preparing libraries from total RNA for RNA sequencing, gene expression analysis and transcriptomics.

16S V4 Amplicon

The library is prepared using a single PCR amplification from genomic DNA samples, using Illumina tailed single indexed primers which bind to the regions flanking the V4 hyper-variable region of 16S rRNA. Up to 48 samples can be multiplexed onto one Illumina MiSeq run. In most microbial species, the V4 hyper-variable region of 16S rRNA is approximately 254 bp, and only deviates from this length by a few base pairs. With the Illumina sequencing both strands of this 254 bp region are sequenced using a 150 bp read.

16S V3-V4 Amplicon

The library is prepared using a single PCR amplification from genomic DNA samples, using Illumina tailed single indexed primers which bind to the regions flanking the V3-V4 hyper-variable region of 16S rRNA. Up to 384 samples can be multiplexed onto one Illumina MiSeq run. In most microbial species, the V3-V4 hyper-variable region of 16S rRNA is approximately 460-470 bp in length. With the Illumina sequencing both strands of this 460-470 bp region are sequenced using a either a 250 bp or 300 bp read.

Custom Amplicon

Custom amplicon libraries are prepared using “two step PCR” amplification from genomic DNA samples. The first PCR step amplified the target region of interest, using Illumina tailed primers containing a sequence which is complementary and will bind to the second step PCR primers.  The first step PCR primers should be designed to target a region of ≤ 550 bp.The second PCR step uses the Illumina Nextera dual index primers for PCR amplification, allowing up to 96 samples to be pooled onto one Illumina MiSeq run. With the Illumina sequencing both strands of this ≤550 bp region are sequenced using a either a 250 bp or 300 bp read, depending on the size of the amplicon product.

Massey Contact Centre Mon - Fri 8:30am to 5:00pm 0800 MASSEY (+64 6 350 5701) TXT 5222 contact@massey.ac.nz Web chat Staff Alumni News Māori @ Massey