A robust targeted sequencing approach for low input and variable quality DNA from clinical samples.

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification-this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.

High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Minimizing loss of sequence information in SAGE ditags by modulating the temperature dependent 3' --> 5' exonuclease activity of DNA polymerases on 3'-terminal isoheptyl amino groups.

Numerous steps are required to prepare a sequencing library for serial analysis of gene expression (or SAGE) from an original mRNA sample. The presence of inefficiencies, however, can lead to a cumulative loss of sample during processing which can yield a library of short sequence tags (SSTs) that represents only a minute fraction of the original starting sample, potentially compromising the quality of the analysis and necessitating relatively large amounts of starting material. We show here that commonly observed higher molecular weight (HMW) amplification products observed following the PCR amplification of ditags are a direct result of the presence of HMW ligation products created during ditag formation. Using model tags, we demonstrate that the formation of these HMW ligation products becomes permissible following the release of the 3'-terminal isoheptyl amine (3'-IHA) from the SST during the fill-in reaction with the Klenow fragment (KF) of DNA polymerase (DNAP) I and is mediated by its 3' --> 5' exonuclease activity. We further show that the incorporation of SSTs into HMW ligation products can lead to a loss of sequence information from SAGE analysis, potentially skewing sequencing results away from the true distribution in the original sample. By modifying fill-in conditions through the use of Vent DNAP at 12 degrees C and by including terminal phosphorothioate linkages within the SAGE adaptors to specifically inhibit exonucleolytic removal of the 3'-terminal amine, we are able to maximize the yield of ditags and bypass the need for gel purification via PAGE following PCR. The modifications described here, combined with the modifications described previously by our group for adaptor ligation, ensure that the full sequence information content in SSTs derived from the transcriptome is preserved in the pool of amplified ditags prior to the creation of a SAGE library.

Increasing the efficiency of SAGE adaptor ligation by directed ligation chemistry.

The ability of Serial Analysis of Gene Expression (SAGE) to provide a quantitative picture of global gene expression relies not only on the depth and accuracy of sequencing into the SAGE library, but also on the efficiency of each step required to generate the SAGE library from the starting mRNA material. The first critical step is the ligation of adaptors containing a Type IIS recognition sequence to the anchored 3' end cDNA population that permits the release of short sequence tags (SSTs) from defined sites within the 3' end of each transcript. Using an in vitro transcript as a template, we observed that only a small fraction of anchored 3' end cDNA are successfully ligated with added SAGE adaptors under typical reaction conditions currently used in the SAGE protocol. Although the introduction of approximately 500-fold molar excess of adaptor or the inclusion of 15% (w/v) PEG-8000 increased the yield of the adaptor-modified product, complete conversion to the desired adaptor:cDNA hetero-ligation product is not achieved. An alternative method of ligation, termed as directed ligation, is described which exploits a favourable mass-action condition created by the presence of NlaIII during ligation in combination with a novel SAGE adaptor containing a methylated base within the ligation site. Using this strategy, we were able to achieve near complete conversion of the anchored 3' end cDNA into the desired adaptor-modified product. This new protocol therefore greatly increases the probability that a SST will be generated from every transcript, greatly enhancing the fidelity of SAGE. Directed ligation also provides a powerful means to achieve near-complete ligation of any appropriately designed adaptor to its respective target.