Sequencing of circulating nucleic acids from low serum volumes

Circulating nucleic acids released from tissues have been found to harbor tumor-specific molecular alterations and to be representative for the molecular composition of the tumor. Next generation sequencing (NGS) of circulating nucleic acids provides the benefits of exome-wide information and allows the identification of novel biomarkers for minimal-invasive cancer diagnostics.

We implemented a workflow for the NGS analysis of circulating nucleic acids from low amounts (200 µl) of serum. First, protocols for isolation, quantification, and QC of nucleic acids, as well as library preparation for exome-enriched DNA or total RNA, including miRNA sequencing, were established. Subsequently, purified DNA and RNA from serum and matched tumor tissue samples from six non-small cell lung cancer patients and from four control sera was sequenced using the Illumina HiSeq technology. Data quality and counts of mappable reads from circulating RNA allowed for transcriptome analysis. Furthermore, the alignment of sequenced reads illustrated different compositions of serum and tissue RNA. Besides fragmented coding transcripts, we found a broad spectrum of non-coding, mitochondrial as well as exogenous RNAs in serum samples. Initial analyses on differential abundance of non-coding RNAs between tissue and serum revealed an accumulation of certain non-coding RNAs in serum samples, indicating a higher stability of these RNA species in the blood.

In addition, we sequenced the exomes, including UTRs of circulating DNA with an average coverage of 146X. Sequence reads of circulating DNA were evenly distributed across the exome and almost equal between serum and tissue.

The established technologies provide the experimental basis for minimal-invasive therapy monitoring as well as the identification on novel blood-based biomarkers on exome and transcriptome level.

*Presenting author