Illumina NGS

Illumina Sequencing

The core offers several flavors of Illumina next generation sequencing.

  • Low-output sequencing is performed on the MiSeq where less than 25 million reads is sufficient for the project. Low-output sequencing is often used to check libraries or for targeted sequencing experiments when high coverage for a few regions is required.
  • Mid-output sequencing is performed on the NextSeq or the smaller (SP, S1, and S2) NovaSeq flow cells. Although more expensive than high-output sequencing, you are purchasing an entire flow cell so turnaround time is faster.
  • High-output sequencing is performed on the larger S4) NovaSeq flow cells. You can purchase 2.5% increments of the 300 cycle flow cell, which means you will be combining with other projects and will need to wait for a flow cell to fill before running. High-output sequencing is the most cost effective sequencing.

Run configurations and expected yield are shown in the table below. These specifications are based on a control library, Illumina PhiX. Actual yields will vary depending on the library type, the library complexity, the insert size, and the GC content. Yields similar to those depicted can be expected for high complexity libraries (e.g. whole genome or standard input RNA-seq libraries). The core makes every effort to target the reads listed ± 20% for all runs.

 

MiSeq Flow Cells ~Number of Reads Cycles Default Run Type
Nano
1 Million Reads
300 PE150
500 PE250
Micro 4 Million Reads 300 PE150
V2
13 Million Reads
50 SR50
300 PE150
500 PE250
V3
25 Million Reads
150 PE75
600 PE300
       
NextSeq 2000 Flow Cells ~Number of Reads Cycles Default Run Type
P1 100 Million Reads 300 PE150
P2
400 Million Reads
100 PE50
200 PE100
300 PE150
P3
1.2 Billion Reads
50 SR50
100 PE50
200 PE100
300 PE150
       
NovaSeq 6000 Flow Cells ~Number of Reads Cycles Default Run Type
SP
800 Million Reads
100 PE50
200 PE100
300 PE150
500 PE250
S1
1.8 Billion Reads
100 PE50
200 PE100
300 PE150
S2
4 Billion Reads
100 PE50
200 PE100
300 PE150
S4
8-10 Billion Reads
200 PE100
300 PE150

 

Illumina Library Prep

Whole Genome Sequencing (WGS)

For WGS, the core supports different options, including standard DNA and PCR-Free. Standard DNA preps are ideal for large genome sequencing when the amount of available DNA is limited. The PCR-Free option enhances the ability to sequence through more challenging genomic regions but requires at least 1ug of intact genomic DNA.

Kits:

  • Standard WGS – NEBNext Ultra II FS DNA Library Prep Kit (NEB, E7805)
  • High-throughput WGS -plexWell High Throughput DNA (seqWell, PW384) 
Illumina Assay Input Range Max Volume Concentration Minimum
Standard Whole Genome 10 – 500ng 35 µl 4.0 ng/µl
High Throughput Whole Genome 3 – 30ng 10 µl 0.3.0 ng/µl

 

Whole Exome Sequencing (WES)

Whole-exome sequencing uses biotinylated oligonucleotide probes to capture only the protein-coding regions (gene exons) from the genomic DNA library. Unlike WGS, only the protein-coding genes are sequenced thereby reducing the amount of sequence needed per sample. Our preferred WES option uses the Swift/IDT Exome Research Panel, but we do work with other probe sets when requested.

Kits:

  • 2S Turbo Flexible DNA (Swift Biosciences, 45096)
  • Swift Exome Hyb Panel (Swift Biosciences, 83216)
Illumina Assay Min Recommended Input Volume Concentration Minimum
Human Whole Exome 100 ng 25 µl 4.0 ng/µl

 

Targeted Sequencing

Targeted sequencing is used when deep sequencing depth over specific genomic regions is desired. There are two methodologies for approaching targeted sequencing assays: probe-based capture sequencing or amplicon sequencing. We can help you select a commercially available panel or design a custom solution to interrogate your genes/regions of interest. Please contact the core for more details. Please note input requirements will depend on your custom assay.

Other DNA

Although we do not perform ChIP or 3C/4C/5C/HiC assays in the core, we do prepare the Illumina libraries from DNA obtained from these and other protocols. Please contact the core to inquire about prep options.

Kits:

  • NEBNext Ultra II DNA Library Prep Kit (NEB, E7645)
Illumina Assay Min Recommended Input Volume Quality and Concentration Minimums
DNA (low input) Library Prep 0.5 ng 25 µl 0.02 ng/µl

 

mRNA-Seq

Messenger RNA accounts for <5% of the RNA in cells. Thus, for transcriptome or gene expression studies, enrichment of samples for mRNAs is preferable. Poly-A selection, in which poly-adenylated transcripts are selected via hybridization to poly-T oligos bound to magnetic beads, is the most commonly used method for enriching mRNA from total RNA samples. For low input (<200ng) samples, the Smart-Seq method starts by hybridizing the RNA to an oligo(dT)-containing primer and then uses a template-switching mechanism to generate full-length cDNA. Both of these polyA-dependent methods generate the highest percentage of reads mapping to protein-encoding genes and thus are the first choice for many investigators. Poly-A enrichment requires high-quality total RNA samples so it is not suitable for FFPE or otherwise degraded samples. 

Kits:

  • Standard – NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and xGen Broad-range RNA Library Prep (IDT, 1009813) with xGen Normalase UDI Primers (IDT, various)
  • Low input – SMART-Seq v4 PLUS Kit (Takara, R400753)
Illumina Assay Min Recommended Input Volume Quality and Concentration Minimums
Poly-A (standard) Library Prep 50 ng 25 µl RIN > 7 and 1 ng/µl
Poly-A (low input) Library Prep 1 ng 25 µl RIN > 7 and 0.095 ng/µl

 

Total RNA-Seq

Ribosomal RNA depletion is applied when transcripts do not carry polyA (e.g. bacterial RNA), when you want to characterize long non-coding RNA (lncRNA) as well as mRNA, or when your RNA is degraded. There are two methodologies for removing ribosomal RNAs (rRNA) from your total RNA. The more common option is a subtractive hybridization method. Suitable for samples with RINs>5, this method uses oligonucleotide probes and magnetic beads to capture and remove the rRNA from the sample. More appropriate for degraded samples (RINs<5) is the RNaseH method which enzymatically degrades rRNA targeted by complementary oligonucleotides.

Kits:

  • Standard H/M/R – NEBNext rRNA Depletion Kit (NEB, E6310) and xGen Broad-range RNA Library Prep (IDT, 1009813) with xGen Normalase UDI Primers (IDT, various)
  • Low input – SMART-Seq Stranded Kit (Takara, 634444)
  • Bacterial – NEBNext rRNA Depletion Kit (NEB, E7850) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760)
Illumina Assay Min Recommended Input Volume Quality and Concentration Minimums
Total RNA (standard) Library Prep 10-40 ng 25 µl

DV200 >70%  – 0.83 ng/µl (10 ng total)

DV200 50-70% – 1.66 ng/µl (20 ng total)

DV200 30-50% – 3.32 ng/µl (40 ng total)

DV200 < 30% not recommended

Total RNA (low input) Library Prep 1-4 ng 25 µl

DV200 >70%  – 0.14 ng/µl (1 ng total)

DV200 50-70% – 0.28 ng/µl (2 ng total)

DV200 30-50% – 0.57 ng/µl (4 ng total)

DV200 < 30% not recommended

 

Small RNA

Due to the size, small RNA is lost during most library prep procedures. For small (micro) RNA, we offer library construction using the SMARTer smRNA-Seq Kit . Small RNA preps can start from either enriched small RNA or directly from total RNA (RIN>7). The minimum amount of total RNA is 100ng, but we recommend starting 500ng-1000ng if possible as the fraction of small RNA in a sample can vary between tissues/organisms.

Kits:

  • SMARTer smRNA-Seq Kit (Takara, 635030)
Illumina Assay Min Recommended Input Volume Quality and Concentration Minimums
small RNA Library Prep 10 ng 15 µl if submitting total RNA RIN > 7

 

 

Frequently Asked Questions 

 

Can I share an Illumina flow cell?

Absolutely! In fact, the most efficient way for the core operate is to get as many projects on S4 flow cells as we can. This is the Core’s standard operating procedure: we strive to fill up 300 cycle S4 flow cells and get them running ASAP.

How long do I have to wait if I am on a NovaSeq S4 shared flow cell?

The Core strives to fill flow cells as quickly as possible. We run two to six flow cells per week on the NovaSeq. The turn-around time for your samples depends on a number of factors, including the quality of your samples (we often get replacements for low-quality samples) and the sample queue length at the time your submission is ready to run. Generally, we should be able to go from “qc passed” to your data in about 4-6 weeks when you share a 300-cycle S4 flow cell. If you want to try to get your data faster, you can submit with low- or mid-output sequencing options to get 100% of a flow cell.

What if my submission for Illumina Sequencing doesn’t need PE150 reads?

Sequencing more bases than needed will not prevent you from doing your experiment. For example, you can trim away uninformative bases. Many users find that the economy of scale makes it cheaper and faster to get longer reads on NovaSeq S4 flow cell than to run the exact required configuration as a previous experiment. Alternatively, if you can fill an entire flow cell yourself, we can work with you to configure a NovaSeq run to your specifications.

What is the minimal increment of a NovaSeq S4 shared flow cell that I can purchase?

The minimal fraction of a 300 cycle S4 flow cell available for purchase is 2.5%, which corresponds to ~200-250 million reads.

Can I see the sample QC reports before library prep starts?

Yes, the Advanced Genomics core will post your QC metrics (BioA traces, concentrations, RINs etc.) in the attachment section of your MiCores request prior to library prep. If the quality and quantity of your samples do not meet the requirements for standard library prep methods, our highly trained staff can provide you with prep alternatives that would accommodate your samples. You will need to make a decision on how to move forward with your samples.

Can I submit replacement samples?

If after initial QC you decide to submit a replacement sample, please contact the core via your MiCores request so additional samples can be added to the request.

Which indexing scheme should I use for Illumina sequencing?

If you prepare your own libraries, we strongly recommend that you dual index your samples to maximize the uniqueness of your sample identifiers. When possible, the core prefers unique dual indices to minimize index hopping. Please contact us if you have specific indexing questions.

What information about my user-made Illumina libraries should I provide during submission?

If you are submitting Illumina-ready libraries, please indicate the type of prep or the kit used to make your libraries as well as any changes/alterations to the standard protocol. This information helps the core optimize the conditions in which your libraries are sequenced.

Do I need to remove primer or adapter contamination from my user-made Illumina libraries?

Yes, primer or adapter-dimer contamination can seriously impact your sequencing run. They not only reduce the number of clusters available for your samples, but can lead to problems such as index hopping.

How do I submit custom Illumina sequencing primers?

Custom primers require the purchase of a full flow cell. You need to verify that the primer sequence is compatible with Illumina platforms prior to submission. In the notes section, please indicate if the custom primer interferes with the PhiX sequencing control (added to all runs by the core). The primer should be submitted in a 1.5 mL tube – the tube must contain 30 uL of the custom primer at a concentration of 100 uM. The tube should be labeled with the following: primer concentration, the service request ID, and the read that the primer is for.

What parameters should I select when submitting RNA for bulk RNA sequencing?
RNA Sequencing MethodpolyA-enrichmentribo-depletionsmall       
Starting MaterialDNase-treated Total RNADNase-treated Total RNADNase-treated Total RNA
RNA QualityRIN >7DV200 > 30%RIN >7
Preferred Input200 - 1000ng200 - 1000ng100 - 1000ng
Preferred Volume25ul25ul25ul
Minimum Input1ng1ng10ng
Recommended Minimum Reads/Sample30 million45 million15 million
Recommended Sequencing PlatformNovaSeqNovaSeqNextSeq or NovaSeq
Recommended Run ConfigurationPE150PE150SR50 (can be longer)
Deliverablesfastq files, count matrixfastq files, count matrixfastq files
Estimated per sample cost (assumes 12 sample submission)$274$389$287
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