Genomic and transcriptomic sequencing

• Illumina Sequencing Coverage Calculator
• Illumina NGS Methods Guide

Extract your RNA with preferred kit (e.g. Qiagens RNeasy Mini Kit (Catalog no. 74104) with Optional On-Column DNase Digestion with the RNase-Free DNase Set (cat. no. 79254)). Please, be sure that your RNA samples are free from DNA, as this will result in an underestimation of the amount of RNA used.



From extracted RNA, three aliquots should be separated before freezing to avoid extra freezing-thawing cycles. These aliquots need to present the sample-aliquots to be sequenced, so no dilutions, or additional freezings for these.

• First aliquot (preferably 8 µl/sample) is for measuring precise concentration of samples with Qubit-assay.
• The second aliquot (2 µl/sample) is for Bioanalyzer-assay for determining the RNA Integrity Number (RIN)-values.
• The third aliquot (15-20 ul) is used for library preparation.

Please contact to ngs (at) oulu.fi for any questions, or if you have only limited amount of RNA.

The purity and approximate concentration should be measured for all samples (e.g. NanoDrop). The recommended thresholds are:

• 260/280 value 1.7-2, preferably 1.8
• 260/230 value > 1.5, preferably > 1,8

Please send this information (purity/concentration) to ngs (at) oulu.fi, to receive instructions how to proceed.

• Transcriptome sequencing (total RNA and mRNA) with Illumina’s NextSeq550
• Libraries are prepared with strand specific kits (Illumina). The amount of samples per run depends on the application (usually 10 to 40 samples).
• The total RNA concentration and quality is checked before preparation of the libraries for sequencing
• The service includes initiation meeting with the customer
• Data is delivered in FASTQ format
• Additional services include assistance in data analysis
• Contact: katri.pylkas (at) oulu.fi, ngs (at) oulu.fi

• Small RNA sequencing and small-scale transciptome sequencing with Ion Torrent
• Libraries are prepared with Ion Total-RNA Seq v. 2.0 kit from Thermo Scientific
• The total RNA concentration and quality is checked before preparation of the RNA libraries for sequencing
• The service includes initiation meeting with the customer
• Data is delivered in FASTQ format
• Additional services: assistance in Data analysis
• Contact: marko.suokas (at) oulu.fi, ngs (at) oulu.fi

• DNA samples are sequenced up to 12 sample batches with Illumina NextSeq550 instrument
• Customer delivers purified DNA samples.
• Total DNA concentration and quality is checked before preparation of the libraries for sequencing
• The service includes initiation meeting with the customer
• Data is delivered in FASTQ and aligned (h19) format
• Additional services include assistance in data analysis
• Contact: katri.pylkas (at) oulu.fi, ngs (at) oulu.fi

• Libraries can be prepared for various gene panels utilizing Nextera rapid capture chemistry
• Customer delivers purified DNA samples.
• Total DNA concentration and quality is checked before preparation of the libraries for sequencing
• The service includes initiation meeting with the customer
• Data is delivered in FASTQ and aligned (h19) format (optional)
• Additional services include assistance in data analysis
• katri.pylkas (at) oulu.fi, ngs (at) oulu.fi

• NextSeq550 has two flow cell configurations with following sequencing performance:
  

  Flow Cell Configuration	Read Length (bp)	Output (Gb)
  High Output Flow Cell*	2 x 150	100-120
  	2 x 75	50-60
  	1 x 75	25-30
  Mid Output Flow Cell**	2 x 150	32-39
  	2 x 75	16-19

* Up to 400 M single reads, and up to 800 M paired-end reads

** Up to 130 M single reads, and up to 260 M paired-end reads

• For details, contact: ngs (at) oulu.fi

Oxford NanoporeTM sequencing technology deliver fast, affordable and flexible sequencing with unparalleled read lengths. You can read about sequencing technology more at www.nanoporetech.com.

Using high accuracy V14 kits, V14 minion flow-cells and sup basecalling allow modal accuracy of >Q20 or 99 % single read accuracy. With additional read filtering, it is possble to achieve median accuracies up to Q35.

Number of reads and sequencing throughput is dependent on library length, quality and sequencing time. It is possible to achieve 20 Gbp raw data. Live basecalling allows flexible sequencing throughputs.

Amplicon sequencing

Our sequencing service focuses to amplicon sequencing with custom dual-indexing option. Amplicons are prepared with two-step PCR protocol, in which in first reaction target DNA is amplified with gene specific primers containing tail sequences. Second PCR will add sample specific indexes to both ends. Nanopore sequencing adapters are ligated to indexed amplicon pool and the library is sequenced on minion flow-cell.

Schematic presentation of indexed amplicon product:

| T2F | Index1 | Tail1 | PrimerF | Region of Interest | PrimerR | Tail2 | Index2 | T2R |

Service also includes support for demultiplexing and trimming sequencing reds.

Other sequencing

As ONT platform is not limited by specific read length, it is possible to sequence also libraries prepared for other platforms. For more information contact: marko.suokas (at) oulu.fi, ngs (at) oulu.fi

Please note that currently, we are not carrying extensive selection of ONT specific library kits or barcoded adapter selection.

More information

For examples of nanopore and other sequencing projects including data analysis check

https://github.com/msuokas-bco