SAMPLE PREPARATION, QUANTIFICATION & QUALITY REQUIREMENTS

 

Illumina MiSeq™ Sequencing APPLICATIONS

Application

Type of Sample to Supply

Illumina MiSeq™ Read lengths recommendations

Method of Library Preparation

Minimum Amount Required

Minimum Concentration Required

Buffer

Whole Genomic DNA Sequencing (WGS) of small genomes (De novo assembly)

Fungal, bacterial, viral and smaller complex genomes

Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)!

Illumina TruSeq™ DNA PCR-Free Library Preparation

1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert)

20ng/µl

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

Whole Genomic DNA Sequencing of small genomes (Re-sequencing)

Fungal, bacterial, viral and smaller complex genomes

Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)!

Illumina TruSeq™ DNA PCR-Free Library Preparation

1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert)

20ng/µl

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

Illumina TruSeq™ DNA Nano Library Preparation

1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert)

20ng/µl

Illumina Nextera™ DNA Library Preparation

100ng DNA

10ng/µl

Illumina Nextera XT™ DNA Library Preparation

1.5ng DNA

0.2ng/ µl

Whole Metagenomic Sequencing

Microbial environmental samples containing a population of microbes

Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)!

Illumina TruSeq™ DNA PCR-Free Library Preparation

1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert)

20ng/µl

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

Illumina TruSeq™ DNA Nano Library Preparation

1.5µg DNA (350 bp insert) or 2.5µg DNA (550 bp insert)

20ng/µl

Illumina Nextera™ DNA Library Preparation

100ng DNA

10ng/µl

Illumina Nextera XT™ DNA Library Preparation

1.5ng DNA

0.2ng/ µl

PCR Amplicon Sequencing

Large Amplicons ≥ 2Kb

Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)!

Illumina Nextera™ DNA Library Preparation

100ng DNA

10ng/µl

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

Illumina Nextera XT™ DNA Library Preparation

1.5ng DNA

0.2ng/ µl

Custom Amplicon Sequencing

Amplicons ≤ 550bp

Illumina MiSeq™ V2 (2x 150 base PE)* or Illumina MiSeq™ V2 (2x 250 base PE)* or Illumina MiSeq™ V3 (2x 300 base PE)! (Run length is dependent on the size of the amplicons being sequenced)

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)

25µL of the amplicon generated from the first PCR step

≥ 1.0ng/µl

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

Metagenomic 16S Amplicon Sequencing V3-V4 hypervariable region

Genomic DNA or PCR Amplicon containing the 16S gene (Amplicon size = ~459 bp)

Illumina MiSeq™ V2 (2x 250 base PE)*

16S V3-V4 Dual-Index Sequencing on the MiSeq Illumina Sequencing Platform#

5-50ng DNA

5ng/µl

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

RNA Sequencing Gene Expression Analysis (Transcriptomics)

Total RNA

Illumina MiSeq™ V2 (2x 150 base PE)* or Illumina MiSeq™ V3 (2x 75 base PE)!

Illumina TruSeq™ Stranded Total RNA Library Preparation

1.5µg Total RNA

200ng/µl

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

Illumina TruSeq™ Stranded mRNA Library Preparation

1.5 µg Total RNA

200ng/µl

NOTES:

* Version 2 Illumina MiSeq™ chemistry. Refer to “Illumina MiSeq™ Run Options” for details.

! Version 3 Illumina MiSeq™ chemistry. Refer to “Illumina MiSeq™ Run Options” for details.

# Reference: http://aem.asm.org/content/79/17/5112

 

IMPORTANT NOTES:

Preparing samples ready for the Illumina Nextera™ DNA Library preparation or 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 ™ DNA Library preparation and Illumina Nextera™ XT DNA Library preparation methods.

 

The minimum product size for the Illumina Nextera™ XT DNA Library preparation is 300 bp. This is to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50 bp from each distal end of a fragment may be seen. This is because the transposon tagmentation enzymatic reaction used to randomly fragment the product, cannot add an adapter right at the distal end of a fragment. This enzymatic clipping of PCR primers avoids wasted sequencing output on non-informative bases that do not contain genomic inserts. If you wish to sequence the genomic loci contained within a PCR primer, simply design your amplicons to be ~100 bases larger than the desired insert to be sequenced.

 

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 library preparation and 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.

 

Alternatively, MGS provide a LabChip Service. Refer to the MGS website for details. For the LabChip Service clients can provide Genomic DNA or PCR Amplicons to the MGS and the service will run them using the PerkinElmer GX Touch HT Instrument using:

  • DNA High Sensitivity Assay – PCR Amplicons
  • Genomic DNA Assay – Genomic DNA

 

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 e.g. Qubit concentration readings and spectrophotometer OD260/280 ratio and OD260/230 ratio readings.

 

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.

Also, the spectrophotometer OD260/280 ratio should be 1.8-2.0; OD260/230 ratio should be 2.0-2.2

 

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.

 

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.

 

Alternatively, MGS provide a LabChip Service. Refer to the MGS website for details. For the LabChip Service clients can provide Total RNA or mRNA to the MGS and the service will run them using the PerkinElmer GX Touch HT Instrument using the Standard RNA Assay.

 

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.

 

The client must supply MGS with an electronic copy of the formaldehyde agarose gel image, or summary report from the bioanalyzer, and the fluorometer e.g. Qubit concentration readings and spectrophotometer OD260/280 ratio and OD260/230 ratio readings.

 

Quality Standards

Bioanalyzer Trace: An RIN or RQS 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; OD260/230 ratio should be 2.0-2.2

 

 

Client Prepared Libraries

MGS will accept client prepared libraries using Illumina compatiable library preparation methods. However, an MGS disclaimer will be included in the MGS Project Plan, 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.

 

The client must supply MGS with the summary report from the bioanalyzer.

 

Quality Standards for Libraries prepared using Illumina Kits

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.

~570-600 bp for libraries prepared for 16S amplicon sequencing of the V3-V4 rRNA hyper-variable region.

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.

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