Biomedicum Genomics Oy
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Next-Generation Sequencing

The Illumina Genome Analyzer IIx sequencing instrumentation, utilizing Illumina's massively parallel sequencing-by-synthesis technology, generates billions of bases of high-quality sequence in a single run. Yielding very low cost per base sequenced, this technology is well-suited for a range of genome-scale applications, such as whole-genome sequencing and resequencing, gene expression profiling (RNA-seq), discovery and detection of small RNAs and mapping of transcription factor binding sites (ChIP-seq). Next-generation sequencing analysis contains four individual steps, which are sample preparation, target amplification / cluster generation, sequencing and data analysis. During sample preparation, samples are fragmented, (cDNA synthesized for mRNA-Seq), DNA-end modified and adapter ligated. Adapter ligated sequences are then hybridized and amplified on glass slide, called flow cell, and these amplified sequences/clusters are sequenced by-synthesis on the Illumina Genome Analyzer IIx. Typically, the experiment data is analyzed using Illumina’s Genome Analysis Pipeline software and customers have the option to retrieve analysis results at any, or all, of the three steps involved in the analysis process, which consists of: Image analysis Base calling Sequence analysis Primary analysis of the raw data, consisting of image analysis, base calling and sequence alignment, are performed using Illumina's Genome Analyzer Pipeline Software. A variety of software packages are employed for application-specific downstream analyses. Targeted Sequence Capture Using Nimblegen Sequence Capture Arrays the customer can focus on specific genomic regions and gain excellent coverage in a single run. First steps include the hybridization and elution of the target enriched sequences from the array. The sample is then amplified and array capture efficiency of each sample is evaluated using qPCR. Captured sequences are then ready for the next-generation sequencing using GAIIx. Prior to array hybridization, samples for sequence capture need to be prepared according to the standard library preparation protocol for next generation sequencing.