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2013|14 Annual Report Fraunhofer IGB

72 FUNCTIONAL GENOMICS VIA NEXT-GENERATION SEQUENCING Dipl.-Biol. Christian Grumaz, Dr. rer. nat. Kai Sohn Sequencing technologies – the next generation The human genome was first decoded in 2001 after 10 years of work by over 100 scientists and a cost of 3 billion US dol- lars [1, 2]. Today, the enormous advancements in sequenc- ing technologies over the most recent years enable a single researcher to decode a human genome within days and for under 10,000 US dollars. The special feature of the technol- ogy referred to as next-generation sequencing (NGS) [3] is the possibility to sequence hundreds of millions of fragments at the same time, as opposed to just individual fragments. These high-throughput or parallel sequencing technologies have opened up entirely new dimensions in nucleic acid analysis and revolutionized countless areas of research in Life Sciences – from de novo genome sequencing up to the early diagnosis of tumor tissue [4]. And the discovery of novel, innovative areas of application has only just begun. The technology at a glance In order to be able to use next-generation sequencing the samples must be processed differently depending on the starting material and purpose of the research. For example, genomic DNA from unknown organisms is used for de novo genome sequencing while a variety of RNA populations (mRNA, small RNA, ncRNA) can be examined in transcriptome analyses. Several sample preparation protocols can also be fully automated using the Biomek FX laboratory automation workstation (Beckman Coulter) at the Fraunhofer IGB. Once a (c)DNA library has been completed it is sequenced either on the Illumina HiSeq2500, with very high read depth and shorter fragments (up to 2 x 100 bases) or by using the Roche GSjunior, which has far lower read depth but can process longer sequences (up to 400 bases). The raw sequencing data can finally be subjected to bioinformatics analysis for the most varied questions, with the aid of the IT infrastructure opti- mized for NGS at the IGB. We have hereby established a three-step process that encom- passes the various steps in sample preparation and sequen­cing in the laboratory, as well as subsequent bioinformatic analysis. The now extremely comprehensive selection of sample prepa- ration protocols and analysis strategies then opens up areas of application, extending from sequencing human genomes with a focus on early detection of cancer, to de novo transcriptome sequencing of biotechnologically relevant production strains or human pathogens, through to the detailed identification of complex biocenotic bacterial populations (metagenomes). Examples of these are briefly presented below. Non-coding RNA as biomarkers The aim of the Fraunhofer project RIBOLUTION is the iden- tification of novel diagnostic indicators for diseases such as COPD and prostate cancer. This involves sequencing of the whole non-coding RNA (ncRNA) population present in blood, a still largely uncharacterized class of molecules. We suspect that they play a decisive role in disease development and therefore have especially great potential as diagnostic bio- markers. 1 applications ¡¡ genomes (de novo, resequencing) ¡¡ transcriptomes (mRNA, small RNA, ncRNA) ¡¡ metagenomes technologies ¡¡ HiSeq2500 (1.6 billion sequences, 2 × 150 bp read length) ¡¡ GSjunior (100,000 sequences, 500 bp read length) analyses ¡¡ de novo assembly ¡¡ reference mapping ¡¡ SNP detection ¡¡ transcriptome annotations ¡¡ gene expression ¡¡ metagenomics MEDICINE

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