Amplicon sequencing - flexible study design combined with high throughput sequencing

Highly sensitive variant detection

Your amplicon sequencing project

Detection of rare and common SNPs, insertions and deletions to study genetic variations in complex and highly variant samples. Phylogenetic characterization of complex bacterial communities and viral subtyping.

Strategy

Locus-specific amplification and following ultra-deep sequencing of small target regions allow to identify rare and common mutations and hence to fully characterize them.

During sample preparation single amplicons (PCR products) within a mixture of amplicons are clonally amplified by emulsion PCR. This enables the sequencing of individual PCR products without laborious upfront cloning of the target sequences into bacteria. Therefore the Roche GS FLX+ system is perfectly suited for detecting rare genetic variants (detection limits of < 1%) in highly complex and heterogeneous samples. Furthermore the long read length allows covering several SNPs with one read which enables the assignment of haplotypes.

Flexible sample preparation and multiplexing options allow the in-depth analyses of a statistically relevant numbers of samples.

Starting material

• Genomic DNA
• PCR products
• PCR products with specific GS FLX+ Titanium adapter sequences attached (“ready to load” )

Library preparation

Adapter integration through locus specific PCR with GS FLX+ fusion primers

Sequencing

•    Roche GS FLX+ | single reads
•    Coverage depending on required sensitivity of variant detection

Bioinformatics

• Raw data (fasta and fastq)
• Flowgrams (sff)
• Mapping file (ace)
• Consensus sequence (fasta)
• Mapping report (txt)
• Table of all SNPs (tsv)

• Pre-Sequencing
• Single Reads
• Genomes
• Targeted (re)-sequencing
  • > Enrichment
  • > Amplicons
• Human Sample Sequencing
• Transcriptomes
• Metagenome
• Regulome

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