Next Generation Sequencing at BEA

BEA is offering services for different sequencing applications with massively parallel high throughput sequencing on the Illumina platform using the Miseq, Nextseq and Hiseq instruments. The output are billions of bases of data per run which support a wide variety of applications. Samples prepared for sequencing can be indexed (barcoded)and sequenced in single or paired end reads. Services at BEA include library preparation, quality control, sequencing, and data analysis for different experimental NGS protocols including different RNA and DNA sequencing applications.

For most medium size sequencing projects at BEA we try to use the cost effective Nextseq 550 and 2000 instrument which generates > 400 million reads per flowcell and run. For more focused applications such as targeted gene sequencing, metagenomics, small genome sequencing, targeted gene expression and amplicon sequencing we are using the Illumina MiSeq which generates up to 25 million reads per run and flowcell. Larger sequencing projects can also be handled at BEA and we will help to find the best solution and access to the most appropiate sequencing platforms available.

It is our ambition to fulfill your sequencing need as soon as possible. If you already have your own pooled sequencing libraries, sequencing services at BEA is handled faster. The waiting time can be shortened if you have samples to fill up an entire flowcell on the Nextseq or Hiseq instruments.

System Overview

Illumina Nextseq 2000

The NextSeq 2000 Sequencing system contains the latest instrument design to miniaturize the volume of the sequencing reaction while increasing output and reducing the cost per run. The NextSeq 2000 uses patterned flow cells and a novel resolution optics system that yields highly accurate imaging data higher sensitivity than previous systems. Different output reagents sizes are available from 50-300 cycles generating >1000 Mreads/run.

Illumina Nextseq 550

Fast and flexible sequencer enables transcriptome and targeted resequencing applications for various project sizes and sequencing throughputs. Nextseq 550 reagent kits with different cycles are available from 75-300 cycles. Libraries are loaded into one flowcell and sequencing data are generated in 12 hours for a 75 cycle sequencing run. The output size makes the Nextseq suitable to analyse 20-40 gene expression profiling samples or 10-20 ChIP sequencing samples.

Illumina MiSeq

Variable read length from 50 to 600 cycles, single read (SR) or paired end (PE). 1 flowcell (1 lane) generates up to 25 million reads per run in SR mode. System is ideal for focused sequencing, smallRNA-Seq, library QC, prokaryotic samples, small genome sequencing and targeted capture.

Illumina HiSeq 2000

Read length: 50 or 100 bp in single read or paired end mode. Run time between 2.5-8 days. Depending on target concentration and cluster generation the data output for 1 flowcell (8 separate lanes) produces up to 1.6 billion reads/run which equals up to 200 million reads/lane with the SBS v3 chemistry. The system is ideal for mRNA-Seq, ChIP-seq and large genome sequencing. The HiSeq 2000 allows multiplexing of several samples and can be operated in single or dual flow cell mode independently allowing different read lengths to run simultaneously.

NGS Library Preparation

The success of library preparation depends on the processing of DNA and RNA. DNA or RNA samples are therefore carefully checked for size, integrity, quality and concentration with Agilent Bioanalyzer/Tapestation and Qubit. Please enquire for the sample recommendations for desired specific library protocol. Typical sample requirements for our standard protocols:

mRNA-seqNEBNEB Ultra II Directional RNA Library Prep 10-1000 ng total RNA≤50ul
mRNA-seqILLUMINATruSeq RNA stranded RNA Library Prep 100-1000 ng total RNA≤50ul
Total RNA-seq with rRNA depletionNEBNEB Ultra II Directional RNA Library Prep 10-1000 ng total RNA≤12ul
Total RNA-seq with rRNA depletionILLUMINATruSeq RNA stranded RNA Library Prep 100-1000 ng total RNA≤10ul
DNAseq/ChIp-seqNEBNEB_Ultra II DNA 0.5-10 ng DNA≤50ul
DNAseq/ChIp-seqRubiconThruplex library prep 100 pg-10 ng DNA≤10ul
DNAseq/AmpliconsILLUMINANextera XT DNA 1ng DNA (300 bp)≤5ul
DNAseq/AmpliconsILLUMINANextera XT DNA 1ng DNA (300 bp)≤5ul
Low input RNA seqClontech/TakaraSMART-Seq v4 Ultra Low Input RNA 10 pg-10 ng total RNA or 1-1000 cells in lysis buffer≤10ul
Low input RNA seqQIAgenQIAseq FX Single RNA 50 pg-10 ng total RNA or 1-1000 cells in lysis buffer≤8ul
16S rRNA seqILLUMINA16S Metagenomic Sequencing Library 15 ng DNA≤3ul
Small RNA seqNEBNEBNext Small RNA library Prep 100-1000 ng total RNA≤6ul

NGS Data Output

Questions concerning the NGS service at BEA can be answered by Fredrik Fagerstrom-Billai.