Accurate SNP and mutation detection by targeted custom microarray-based genomic enrichment of short-fragment sequencing libraries.

Microarray-based enrichment of selected genomic loci is a powerful method for genome complexity reduction for next-generation sequencing. Since the vast majority of exons in vertebrate genomes are smaller than 150 nt, we explored the use of short fragment libraries (85-110 bp) to achieve higher enrichment specificity by reducing carryover and adverse effects of flanking intronic sequences. High enrichment specificity (60-75%) was obtained with a relative even base coverage. Up to 98% of the target-sequence was covered more than 20x at an average coverage depth of about 200x. To verify the accuracy of SNP/mutation detection, we evaluated 384 known non-reference SNPs in the targeted regions. At approximately 200x average sequence coverage, we were able to survey 96.4% of 1.69 Mb of genomic sequence with only 4.2% false negative calls, mostly due to low coverage. Using the same settings, a total of 1197 novel candidate variants were detected. Verification experiments revealed only eight false positive calls, indicating an overall false positive rate of less than 1 per approximately 200,000 bp. Taken together, short fragment libraries provide highly efficient and flexible enrichment of exonic targets and yield relatively even base coverage, which facilitates accurate SNP and mutation detection. Raw sequencing data, alignment files and called SNPs have been submitted into GEO database http://www.ncbi.nlm.nih.gov/geo/ with accession number GSE18542.

Intraoperative imaging in pathology-assisted surgery.

The pathological assessment of surgical specimens during surgery can reduce the incidence of positive resection margins, which otherwise can result in additional surgeries or aggressive therapeutic regimens. To improve patient outcomes, intraoperative spectroscopic, fluorescence-based, structural, optoacoustic and radiological imaging techniques are being tested on freshly excised tissue. The specific clinical setting and tumour type largely determine whether endogenous or exogenous contrast is to be detected and whether the tumour specificity of the detected biomarker, image resolution, image-acquisition times or penetration depth are to be prioritized. In this Perspective, we describe current clinical standards for intraoperative tissue analysis and discuss how intraoperative imaging is being implemented. We also discuss potential implementations of intraoperative pathology-assisted surgery for clinical decision-making.

Combined transmission, dark field and fluorescence microscopy for intact, 3D tissue analysis of biopsies.

SIGNIFICANCE: Currently, tissue biopsies are sectioned into 3- to 5-µm-thick slices that are used for conventional pathology analysis. Previous work by confocal microscopy and light-sheet microscopy has shown that analyzing biopsies intact in three-dimensions (3D) is possible and may lead to a better understanding of cancer growth patterns. Although accurate, these methods require fluorescent staining of the tissue, in addition to tissue clearing. If the 3D biopsy analysis could be done sufficiently swiftly, this approach may be used for on-site assessment of the adequacy of a biopsy taken. AIM: We aim to show that, by transmission microscopy of optically cleared tissue punches, the tissue architecture can be determined without the need for fluorescent staining. APPROACH: Transmission microscopy is used by combining bright field microscopy with dark field and epifluorescent microscopy to compare samples that have also been analyzed by fluorescent confocal microscopy. RESULTS: With increasing distance to the focal plane, the higher-frequency part of the spatial frequency spectrum of transmitted light is attenuated increasingly. This property is exploited for tissue segmentation, detecting whether tissue is present at a certain position in the focal plane image. Using this approach, we show that a 3D rendering of the internal cavity or tubules structure of punch biopsies, which are up to 1-mm thick, can be acquired in ≈1 min scan time per imaging modality. The images of the overall tissue architecture that are obtained are similar to those from the confocal microscopy benchmark, without requiring fluorescent staining. CONCLUSIONS: Images of the overall tissue architecture can be obtained from transmission microcopy; they are similar to those from the confocal microscopy benchmark without requiring fluorescent staining. Tissue clearing is still needed. The total scan time of the present method is significantly shorter at a fraction of the device costs.

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device.

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor.