Whole Genome Amplification Kit Research Articles

Cost‐saving population genomic investigation of Daphnia longispina complex resting eggs using whole‐genome amplification and pre‐sequencing screening

Resting stages of aquatic organisms that accumulate in the sediment over time are an exceptional resource that allows direct insights into past populations and addressing evolutionary questions. This is of particular interest in taxa that face relatively new environmental challenges, e.g., climate change and eutrophication, such as the Daphnia longispina species complex, a keystone zooplankton group in European freshwater ecosystems. However, genomic analysis might be challenging as DNA yield from many of these resting stages can be low and the material degraded. To reliably allow the resequencing of single Daphnia resting eggs from different sediment layers and characterize genomic changes through time, we performed whole-genome amplification to obtain DNA amounts suitable for genome resequencing and tested multiple protocols involving egg isolation, whole-genome amplification kits, and library preparation. A pre-sequencing contamination screening was developed, consisting of amplifying mitochondrial Daphnia and bacterial markers, to quickly assess and exclude possibly contaminated samples. In total, we successfully amplified and sequenced nine genomes from Daphnia resting eggs that could be identified as Daphnia longispina species. We analyzed the genome coverage and heterozygosity of these samples to optimize this method for future projects involving population genomic investigation of the resting egg bank.

P-533 Single-cell long-read nanopore sequencing as a fast and cost-efficient method for aneuploidy detection and potentially PGT-A, a pre-clinical study

Abstract Study question Is long-read nanopore sequencing feasible and reproducible as routine technique for aneuploidy detection, potentially allowing fresh embryo transfer PGT-A cycles? Summary answer Pre-clinical analysis of euploid and aneuploid DNA and single cells using long-read nanopore sequencing resulted in high concordance and reproducible results for aneuploidy detection. What is known already PGT-A is mostly performed using short-read next-generation sequencing, which requires high initial investment costs and high running expenses. Third-generation sequencing is a novel sequencing technology with the potential of fast, easy, and cost-effective sequencing analysis, possible even for small and less well-financed clinics. Long-read nanopore sequencing for PGT was mainly shown for structural variants and monogenetic disease, which comes with high costs and is far from clinical routine. PGT-A from trophectoderm biopsy samples using nanopore sequencing was so far demonstrated in a small pilot study and further pre-clinical and clinical studies are needed to transfer the technology into clinical routine. Study design, size, duration In this pre-clinical study, euploid and aneuploid DNA and single cells, as well as three to 20 cells were analyzed for aneuploidy using two different whole genome amplification (WGA) kits and long-read nanopore sequencing. In total, 44 different samples were analyzed after multiple displacement amplification (MDA) using REPLIg WGA kit (QIAGEN) and so far, 15 samples were analyzed using PicoPlex WGA kit (Takara) from April 2021. To confirm reproducibility, certain WGA samples were sequenced repetitively. Participants/materials, setting, methods Different euploid and aneuploid gDNA samples were diluted to 4.5 pg DNA per sample. Four different human fibroblast cell lines were diluted to approx. 20 cells, or single cells/three cells were picked using micromanipulation technique. DNA and cells were amplified, prepared for sequencing, and sequenced on MinION sequencer from Oxford Nanopore Technology. Data were analyzed using a custom pipeline consisting of pre-processing, alignment and copy-number calling. QC values and whole chromosome aneuploidies were determined automatically. Main results and the role of chance From 44 different single cells (n = 14), few cells (n = 9) or diluted gDNA (n = 21) samples amplified using MDA, 42 samples showed good quality sequencing results. Two samples result in QC failure, yielding a sample-success-rate (SSR) of 95.5%. Overall, sample sensitivity was 100%, specificity 95.2% with positive predictive value (PPV) of 95.5% and negative predictive value (NPV) of 100%. Per chromosome sensitivity was 100%, specificity 99.8% with PPV of 93.3% and NPV of 100%. Using PicoPlex WGA, 15 samples were analyzed so far: 7 gDNA and 8 single cell samples. The study is ongoing. All samples analyzed resulted in high quality sequencing data with the correct karyotype, leading to 100% SSR, 100% sensitivity, specificity, PPV, NPV per samples as well as per chromosome. Repetitive sequencing (n = 3) of four MDA amplified single cells showed identical sequencing results, indicating high reproducibility of library preparation, and sequencing. A 5p deletion in one cell line was correctly identified in all single cell analyses from either MDA or PicoPlex amplification, indicating sufficient resolution even for segmental aneuploidies. The whole workflow is feasible in under 24 hours. These results indicate high accuracy and high reproducibility of nanopore sequencing technology for single cell aneuploidy detection, possibly transferable for PGT-A. Limitations, reasons for caution The results of this pre-clinical study look very promising and workflow for sample preparation, sequencing and data analysis is fast, cost-efficient, and feasible for PGT-A applications. Nevertheless, no polar body, blastomere or trophectoderm biopsy sample was used and a clinical trial using real clinical samples is needed to confirm applicability. Wider implications of the findings Implementing novel technologies into clinical routine requires extensive systematic pre-clinical and clinical testing. To our knowledge, this is the first study that systematically analyzes sensitivity, specificity, PPV and NPV for aneuploidy detection using long-read sequencing technology. The fast workflow principally allows day 3/5 fresh embryo transfer after polar body biopsy. Trial registration number Not applicable

Refining the evolutionary time machine: An assessment of whole genome amplification using single historical Daphnia eggs.

Whole genome sequencing is instrumental for the study of genome variation in natural populations, delivering important knowledge on genomic modifications and potential targets of natural selection at the population level. Large dormant eggbanks of aquatic invertebrates such as the keystone herbivore Daphnia, a microcrustacean widespread in freshwater ecosystems, provide detailed sedimentary archives to study genomic processes over centuries. To overcome the problem of limited DNA amounts in single Daphnia dormant eggs, we developed an optimized workflow for whole genome amplification (WGA), yielding sufficient amounts of DNA for downstream whole genome sequencing of individual historical eggs, including polyploid lineages. We compare two WGA kits, applied to recently produced Daphnia magna dormant eggs from laboratory cultures, and to historical dormant eggs of Daphnia pulicaria collected from Arctic lake sediment between 10 and 300years old. Resulting genome coverage breadth in most samples was ~70%, including those from >100-year-old isolates. Sequence read distribution was highly correlated among samples amplified with the same kit, but less correlated between kits. Despite this, a high percentage of genomic positions with single nucleotide polymorphisms in one or more samples (maximum of 74% between kits, and 97% within kits) were recovered at a depth required for genotyping. As a by-product of sequencing we obtained 100% coverage of the mitochondrial genomes even from the oldest isolates (~300years). The mitochondrial DNA provides an additional source for evolutionary studies of these populations. We provide an optimized workflow for WGA followed by whole genome sequencing including steps to minimize exogenous DNA.

Evaluating the effects of whole genome amplification strategies for amplifying trace DNA using capillary electrophoresis and massive parallel sequencing

To draw robust conclusions when trace DNA samples are detected in complex cases, it is essential to successfully recover and genotype short tandem repeats (STRs) from trace DNA. However, obtaining complete STR profiles by the conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) method is generally difficult as trace DNA is often less than 100 pg. Previous studies have proven that through whole-genome amplification (WGA), the yield of DNA from trace DNA samples could be improved. In this study, we used two WGA kits, namely, REPLI-g® Single Cell kit and MALBAC® Single Cell DNA Quick-Amp Kit (hereafter referred to as REPLI and MALBAC), to amplify DNA samples with a series of dilutions (from 5.00 ng/μL to 0.391 pg/μL). Typing of STR markers in samples with and without WGA were then performed on a CE platform by the application of Goldeneye® DNA ID System 20 A kit, as well as directly calling sequences from massive parallel sequencing (MPS) for WGA samples with 1.00 ng, 125 pg and 25.0 pg as DNA inputs. Quantification results demonstrated that the yield of samples with WGA could reach the microgram level. The amplification fold was at least > 2000 and > 200 for REPLI and MALBAC, respectively. CE results showed that the number of correctly called loci was improved for trace DNA after WGA when the DNA inputs were lower than 25.0 pg for REPLI and 6.25 pg for MALBAC, respectively. WGA remarkably improved the percentage of called loci with DNA inputs lower than 50.0 pg, although poor performance in repeatability was observed. MPS results suggested that the correctly called loci calculated by MPS reads were mostly more than those calculated by CE, particularly for those of short length, implying MPS of samples after WGA is worth testing in the future. In conclusion, WGA has the potential usability for forensic trace DNA analysis at the single-cell level with good fidelity, although its repeatability requires further improvement.

Comparison of seven single cell whole genome amplification commercial kits using targeted sequencing

Advances in whole genome amplification (WGA) techniques enable understanding of the genomic sequence at a single cell level. Demand for single cell dedicated WGA kits (scWGA) has led to the development of several commercial kit. To this point, no robust comparison of all available kits was performed. Here, we benchmark an economical assay, comparing all commercially available scWGA kits. Our comparison is based on targeted sequencing of thousands of genomic loci, including highly mutable regions, from a large cohort of human single cells. Using this approach we have demonstrated the superiority of Ampli1 in genome coverage and of RepliG in reduced error rate. In summary, we show that no single kit is optimal across all categories, highlighting the need for a dedicated kit selection in accordance with experimental requirements.

22. A NOVEL, FAST, SINGLE TUBE AMPLIFICATION AND INDEXING APPROACH FOR PGT-A FOR ILLUMINA® SEQUENCING INSTRUMENTS

Introduction Most methods to prepare embryo biopsy samples for Preimplantation Genetic Testing for Aneuploidy (PGT-A) for Illumina® sequencing instruments use a two-step approach of Whole Genome Amplification (WGA) followed by library preparation. A single tube, PCR indexing approach offers several advantages for laboratories performing PGT-A including protocol time efficiencies and reduced hands on time. A novel protocol has been developed based on the PerkinElmer DOPlify® WGA kit. The aim of this study was to determine the accuracy and performance characteristics of this protocol on 5 cell samples from euploid, single and double trisomy cell lines. Material and methods Five-cell samples representative of trophectoderm biopsy were manually sorted from aneuploid cell lines (Coriell Institute) and euploid lymphocytes. Cell lysis and WGA were performed using a modified DOPlify® kit protocol (PerkinElmer) followed by incorporation of Illumina specific adapter sequences and unique indexes in a novel single tube approach. Amplified, indexed cell samples were purified, quantified then pooled before 50 sample multiplex and 1 × 75bp read length sequencing on the Illumina MiSeq® Instrument using v3 chemistry. Sequencing data was analysed for correct aneuploidy calling using the PG-Find™ software (PerkinElmer). Results A total of 104 multicell samples were processed with the following karyotypes: 47,XY,+15 (n=22), 47,XX,+18 (n=20), 48,XXY,+21 (n=20), 46,XY (n=22) and 46,XX (n=20). Three samples were excluded from final analysis due to weak amplification (3%). All other samples were processed through to sequencing and displayed the expected karyotype when analysed with the PG-Find™ software, with no false positives or false negatives observed. The 50-sample multiplex generated an average of 510,000 mapped reads per sample with a 98.9% mapping rate to hg19. The novel protocol took on average 4.5 hours (2.5 hours hands on) to process 50 samples from sample receipt to MiSeq® Instrument loading. Conclusions The novel one tube amplification and indexing method was shown to be a fast, accurate protocol for PGT-A utilising Illumina® sequencing instruments. Sequencing ready, 50 sample multiplex pools were prepared in 4.5 hours, readily enabling a less than 24 hour turn-around time from sample receipt to reporting. Studies processing single cells (representative of blastomere biopsy) and cell samples with smaller segmental aberrations to determine the resolution of the protocol are underway, which will be followed by clinical validation using embryo biopsy samples.

7. GENETIC TESTING FOR COPY NUMBER VARIATIONS IN FIRST POLAR BODIES AND OOCYTES BY SINGLE-CELL NEXT-GENERATION SEQUENCING

Introduction Female meiotic errors are the main cause of whole chromosomal aneuploidy in human embryos and significantly contribute to early embryo loss. Meiotic chromosomal malsegregation occurs during divisions of the maturing oocyte in oogenesis concurrently with extrusion of two polar bodies: polar body first (PB1) and polar body second (PB2). Based on meiosis mechanism, genome of a single oocyte can be deduced by analyzing its sibling polar bodies. PB genetic testing can identify abnormalities of maternal meiotic origin and may be the only form of preimplantation genetic testing for aneuploidies (PGT-A) accepted by patients who object embryo biopsy for ideological reasons. In the majority of previous studies, array comparative genomic hybridization (aCGH) was a method of choice for comprehensive chromosome testing in polar bodies. Here, we test the use of single-cell next-generation sequencing (NGS) method in polar body testing for detecting copy number variations (CNVs) in PB1 genome and validate methods for whole genome amplification (WGA) of PB1. Material and methods In this study, single-cell NGS technology was used to asses ploidy status of oocytes and their sibling PB1. A total of 120 single cells (60 first polar bodies and 60 oocytes) were collected from in vitro fertilization (IVF) couples and subjected for WGA using four different kits based on three distinct amplification systems: (i) polymerase chain reaction (PCR-based WGA), (ii) multiple displacement amplification (MDA) or (iii) multiple annealing and looping-based amplification cycles (MALBAC). WGA products were processed and analyzed with VeriSeq PGS MiSeq kit on MiSeq sequencing platform (Illumina) and random samples were validated with the aCGH method. Amplification uniformity and accuracy for CNV detection of the four tested kits were evaluated. Results Ploidy status was determined for 112 of 120 (93,3%) samples (84/112 based on direct analysis and 28/112 based on indirect analysis of the well-amplified sibling PB1 or oocyte). Estimated aneuploidy rate for the studied group which resembles the total oocyte abnormality resulting from meiosis I errors rate was 33% (37/112 samples). In the group of well-amplified PB1-oocyte pairs (33/60) a 97% concordance between the chromosomal status of PB1 and the corresponding oocyte was observed. Both whole chromosome nondisjunction (6 of 37 aneuploid samples) and abnormal chromatid pre-division and segregation were detected (31 of 37 aneuploid samples). The result is concordant with previous studies - abnormal chromatid segregation was the dominant aneuploidy-causing mechanism. Differences in WGA kits performance including amplification uniformity, aneuploidy calling potential and a number of interpretable NGS results were evaluated and the most optimal kit for PB1 genome WGA was pre-selected. Results from aCGH validation of the randomly selected samples showed full consistency with the NGS results. Conclusions The NGS-based method used for PB1 genome analysis showed a high predictive potential of PB1 in deducing ploidy status of the corresponding oocyte and is a promising method for PGT-A of oocytes. The study also contributes to a better understanding of the chromosome segregation patterns and maternal meiosis errors. Supported by: POIR.02.03.02-14- 0092/17

Preimplantation genetic diagnosis and screening (PGD/S) using a semiconductor sequencing platform

BackgroundRecent advances in semiconductor sequencing platform (SSP) have provided new methods for preimplantation genetic diagnosis/screening (PGD/S). The present study aimed to evaluate the applicability and efficiency of SSP in PGD/S.MethodsThe artificial positive single-cell-like DNAs and normal single-cell samples were chosen to test our semiconductor sequencing platform for preimplantation genetic diagnosis/screening (SSP-PGD/S) method with two widely used whole-genome amplification (WGA) kits. A total of 557 single blastomeres were collected from in vitro fertilization (IVF) couples, and their WGA products were processed and analyzed by our SSP-PGD/S method in comparison with array comparative genomic hybridization (array-CGH).ResultsOur SSP-PGD/S method indicated high compatibilities with two commercial WGA kits. For 557 single blastomeres, our method with four million reads in average could detect 24-chromosome aneuploidies as well as microdeletion/microduplication of the size over 4 Mb, providing 100% consistent conclusion with array-CGH method in the classification of whether it was transplantable.ConclusionsOur studies suggested that SSP-PGD/S represents a valuable alternative to array-CGH and brought PGD/S into a new era of more rapid, accurate, and economic.

Abstract 5601: Single-cell molecular profiling of circulating tumor cells (CTCs) within the TRACERx study reveals heterogeneous patterns in early non-small cell lung cancer (NSCLC)

Abstract Introduction: For early stage NSCLC, surgery with curative intent is the most common therapeutic option; however, tumour recurrence occurs in approximately 50% of cases and most commonly at distant sites. Analysis of CTCs represents a potential means of tracking cancer cells from the primary tumour and has the potential to provide insights into the metastatic process and mechanisms of resistance to therapy linking cell phenotype and genotype. Within the TRACERx study, we are carrying out whole genome analysis of single CTCs obtained from NSCLC patients undergoing surgery in order to examine the relationship between CTCs, the primary tumour and metastatic relapse. Methods: Blood from 163 patients was collected at surgery from the tumour draining pulmonary vein and from peripheral vein in longitudinal samples. CTCs were enriched based on their EpCAM expression using CellSearch® and then enumerated according to their positivity for epithelial markers (Cytokeratins) and lack of detectable blood markers (CD45). Samples containing at least five CellSearch® CTCs (CS-CTCs) were processed through DEPArray™ for single cell isolation followed by whole genome amplification (WGA) using the Ampli1™ WGA kit. Genome wide copy number alterations (CNA) were detected through low-depth whole genome sequencing (WGS) and mutational analysis of individual CTCs was carried out using whole exome sequencing (WES). CTC genomic profiles were compared to the corresponding spatially separated tumour sections obtained at surgery. Results: In a pilot study of 30 patients undergoing tumour resection, the presence of pulmonary vein CS-CTCs has been linked to poor clinical outcome (Crosbie et al, J Thorac Oncol, 2016). Here we extended the evaluation of pulmonary vein CS-CTCs to a further 122 patients and confirmed the initial observation that the presence of pulmonary vein CS-CTCs is a poor prognostic indicator. CNA analysis of 100 single CTCs from the first 12 patients revealed heterogeneous patterns between patients and within individual patients. Three types of pulmonary CS-CTC candidates were observed: Type 1, which share clear copy number changes with both tumour and other CS-CTCs; Type 2, where copy number changes were detected but were not shared with the tumour; Type 3, where no copy number changes were detected. Using WES of both CTCs and matching excised tumour regions, we identified common genetic alterations shared by CTCs and the tumour, as well as private genetic changes detected only in CTCs. We are currently evaluating metastatic biopsies from relapsing patients and comparing their molecular profiles to those seen in CS-CTCs obtained at surgery. Conclusion: The combined tumour and CTC data show that single CTC analysis provides an additional layer of complexity with a valuable new perspective on tumour heterogeneity and early dissemination in NSCLC. Citation Format: Francesca Chemi, Sakshi Gulati, Dominic G. Rothwell, Debbie Burt, Daniel Slan-Tan, Barbara Mesquita, Chris Wirth, Gareth Wilson, Jackie Pierce, Ged Brady, Charles Swanton, Caroline Dive. Single-cell molecular profiling of circulating tumor cells (CTCs) within the TRACERx study reveals heterogeneous patterns in early non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5601.

Comparison of single cell sequencing data between two whole genome amplification methods on two sequencing platforms

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells

To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Unfortunately, studies comparing different WGA methods for downstream human identification Short Tandem Repeat (STR) analysis remain absent. Therefore, the aim of this work was to assess the performance of four commercially available WGA kits for downstream human identification STR profiling on a B-lymphoblastoid cell line. The performance was assessed using an input of one or three micromanipulated cells. REPLI-g showed a very low dropout rate, as it was the only WGA method in this study that could provide a complete STR profile in some of its samples. Although Ampli1, DOPlify and PicoPLEX did not detect all selected STR markers, they seem suitable for genetic identification in single-cell applications.

The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection.

With the development and clinical application of genomics, more and more concern is focused on single-cell sequencing. In the process of single-cell sequencing, whole genome amplification is a key step to enrich sample DNA. Previous studies have compared the performance of different whole genome amplification (WGA) strategies on Illumina sequencing platforms, but there is no related research aimed at Ion Proton platform, which is also a popular next-generation sequencing platform. Here by amplifying cells from six cell lines with different karyotypes, we estimated the data features of four common commercial WGA kits (PicoPLEX WGA Kit, GenomePlex Single Cell Whole Genome Amplification Kit, MALBAC Single Cell Whole Genome Amplification Kit, and REPLI-g Single Cell Kit), including median absolute pairwise difference, uniformity, reproducibility, and fidelity, and examined their performance of copy number variation detection. The results showed that both MALBAC and PicoPLEX could yield high-quality data and had high reproducibility and fidelity; and as for uniformity, PicoPLEX was slightly superior to MALBAC.

Abstract 1724: Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing

Abstract Background: Genomic characterization of circulating tumor cells (CTCs) provides insights into cancer genetic changes, and might be utilized for cancer prognosis, diagnosis, as well as monitoring of therapeutic efficacy. Targeted Panel Next Generation Sequencing (NGS) enables analyzing CTC genetic variants of a focused gene panel at a relatively lower cost1. However, CTCs are rare, often resulting in very limited DNA quantities available that require whole genome amplification (WGA). In previous studies, we introduced the Vortex technology, a platform enabling label-free enrichment of CTCs from blood samples of colorectal cancer (CRC) patients and their use for genomic assays downstream2. In this study, we developed a simple and efficient NGS workflow for CTC samples collected by this technology. Method: An optimized workflow using the Qiagen GeneRead DNAseq targeted panel and Illumina MiSeq NGS was first verified on HCT116 CRC cell line before being applied on patient CTCs. For patient blood samples, CTCs were collected with the Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. Matched white blood cell (WBC) DNA was included to subtract germline background. Fresh frozen liver metastasis tissue was collected and analyzed using the same NGS workflow. DNA from CTCs was extracted and amplified using Qiagen REPLI-g single cell WGA kit. Mutation detection on the WGA amplified DNA was performed using the GeneRead DNAseq CRC targeted panel of 38 genes and MiSeq sequencing. The sequencing data were analyzed by QIAGEN NGS Data Analysis Web Portal and Ingenuity Variant Analysis software. Results: The Vortex technology was validated for the capture of CTCs from CRC patients. REPLI-g performed a uniform, unbiased amplification on fresh rare cells with a coverage of 97.7%, which enabled further targeted panel NGS. Blood from 3 CRC patients (P1, P2, P3) and 2 healthy donors (HD1, HD2) was processed with Vortex platform. Less than 1 CTCs/mL blood were found in HD1 and HD2. P1 and P2 had 66 and 20 CTCs/ mL of blood respectively, with many vimentin positive CTC clusters. P3 had 2 CTCs/mL of blood. No somatic mutation was found in healthy donors. Somatic variants were only detected in the CTCs from patient samples that were not present in matched germline WBCs. For P1, more mutations were found in the CTCs than in the liver metastasis while it was the opposite for P2 and P3. Conclusion: For each patient, variants in CTCs and germline WBCs were analyzed from one blood sample using an optimized targeted NGS workflow and compared to liver mets. Our optimized workflow, using the Qiagen REPLIg and GeneRead DNAseq Targeted Panel NGS enabled the detection of CTC mutations for 38 CRC-focused genes. The inclusion of a germline WBC control in the workflow allowed the detection of mutations from pooled CTC samples collected using the Vortex technology. Altmüller J, et al. (2014). Biol Chem. Kidess-Sigal E, et al. (2016). Oncotarget. Citation Format: Haiyan E. Liu, Melanie Triboulet, Amin Zia, Meghah Vuppalapaty, Evelyn Kidess-Sigal, John Coller, Vanita S. Natu, Vida Shokoohi, James Che, Corinne Renier, Natalie Chan, Violet Hanft, Elodie Sollier-Christen, Stefanie S. Jeffrey. Genomic profiling of Vortex-enriched CTCs using whole genome amplification and multiplex PCR-based targeted next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2017-1724

Abstract 3945: Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing

Abstract Introduction Chromosome instability (CIN) is a hallmark of cancer, acting by boosting genetic alterations responsible of tumorigenesis, progression and heterogeneity. Whole-genome sequencing (WGS) protocols are established methods for studying copy-number alterations (CNA) in single-cells, following a necessary whole-genome amplification (WGA) step. We previously presented a method for single-cell CNA profiling of CTCs based on shallow WGS of LM-PCR based WGA products. Here for the first time, we show that the same method may be employed even on single FFPE cells, overcoming the challenges of DNA degradation and damage linked to this type of samples. Methods Two 50μm thick FFPE sections from pancreas ductal adenocarcinoma with &amp;lt;20% tumor content were processed following DEPArray™ sample preparation protocol, including cell dissociation and staining with vimentin, keratin and DAPI, and DNA quality was assessed using the DEPArray FFPE QC kit (QC=0.7). Cell pools (n=15, range=6-213 cells) and single-cells (n=11) from pure tumor and stromal populations were recovered in separate tubes and lysed. A pool of 86 tumor cells and a pool of 213 stromal cells were used for low-pass WGS on MiSeq, obtaining ≈2M paired-end reads on average. By contrast, other cell pools and single cells were firstly whole-genome amplified using the Ampli1™ WGA kit and then sequenced at shallow coverage using Ampli1 LowPass kit and IonTorrent PGM, with ≈500k single-end reads as mean throughput per library. The genome integrity (GI) of WGA samples was evaluated by Ampli1 QC, a PCR-assay based on 4 amplicons of different lengths. Results The purity of DEPArray sorting was confirmed by the large number of chromosome alterations in sorted tumor and the parallel lack of gains and losses in sorted stromal pools and single-cells. A titration test was conducted measuring the consistency of CNA profiles of tumor WGA products starting from different number of cells, ranging from 117 down to single cells. For the purpose, we employed a Receiver Operating Characteristic (ROC) curve using the non-amplified tumor population as reference. Results showed an excellent performance level with a mean Area Under Curve (AUC) equals to 0.93. Interestingly, lower AUCs (0.87) were observed for single-cells, due to some level of inter-cell heterogeneity. Moreover, the proposed low-pass WGS method demonstrated a high resiliency to DNA degradation as quality of CNA profiles, measured by Derivative Log Ratio Spread (DLRS), only showed a weak correlation with GI level, with high-quality CNA profiles obtained also with the lowest GI value. Conclusions Presented approach for copy-number profiling of tumor single-cells isolated by DEPArray digital sorter and processed with Ampli1 workflow has proven to be a reliable and valuable application for the molecular characterization of tumor clones in degraded samples as FFPE tissues. Citation Format: Alberto Ferrarini, Genny Buson, Chiara Bolognesi, Claudio Forcato, Paola Tononi, Valentina del Monaco, Chiara Mangano, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3945. doi:10.1158/1538-7445.AM2017-3945

Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis.

Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.

Performance of a TthPrimPol-based whole genome amplification kit for copy number alteration detection using massively parallel sequencing.

Starting from only a few cells, current whole genome amplification (WGA) methods provide enough DNA to perform massively parallel sequencing (MPS). Unfortunately, all current WGA methods introduce representation bias which limits detection of copy number aberrations (CNAs) smaller than 3 Mb. A recent WGA method, called TruePrime single cell WGA, uses a recently discovered DNA primase, TthPrimPol, instead of artificial primers to initiate DNA amplification. This method could lead to a lower representation bias, and consequently to a better detection of CNAs. The enzyme requires no complementarity and thus should generate random primers, equally distributed across the genome. The performance of TruePrime WGA was assessed for aneuploidy screening and CNA analysis after MPS, starting from 1, 3 or 5 cells. Although the method looks promising, the single cell TruePrime WGA kit v1 is not suited for high resolution CNA detection after MPS because too much representation bias is introduced.

Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells.

BACKGROUNDWhole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive.RESULTSIn respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most.MATERIALS AND METHODSWe analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms.CONCLUSIONWe demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.

Abstract 3615: Comparison of whole genome amplification methods on single and pooled cells for comparative genomic hybridization array analysis

Abstract Background: Because blood can be easily sampled repeatedly over the course of disease, circulating tumor cells (CTCs) offer an opportunity to measure phenotypic changes during treatment permitting adjustments to therapy as cancer evolves and/or develops resistance. Since cancer therapeutics are often selected based upon molecular information, reliable, high quality methods for whole genome amplification (WGA) are required for single and small pools of CTCs. Here we compared REPLI-g (QIAGEN) and PicoPLEX (Rubicon) WGA methods using picked model CTCs (SKBR3 cells) employing the AccuCyte-CyteFinder-CytePicker system (RareCyte) and subsequent molecular analysis by array comparative genomic hybridization (aCGH). Methods: We compared aCGH performed on WGA products from single and pooled SKBR3 cells processed with the typical AccuCyte protocol, or an alternative protocol using live cells in suspension. For the typical AccuCyte protocol, isolated buffy coat was spread and dried onto microscope slides, fixed and antibody stained (EpCAM, cytokeratin, CD45) on an automated instrument. Alternatively, pure, live SKBR3 cells were stained in suspension for EpCAM and Her2, and then placed into well slides. Slides were imaged using the CyteFinder digital fluorescence scanning microscope. Fixed SKBR cells were identified by positive nuclear, EpCAM, and CK staining, and negative CD45 staining. Live cells were identified by positive EpCAM/Her2 staining. Cells were picked into PCR tubes using the CytePicker module. DNA from individual or pools of cells (2, 3, 5, or 10) was amplified using either the REPLI-g or PicoPLEX WGA kits and processed for aCGH using Genetisure arrays (Agilent). WGA products were hybridized against matched WGA controls from normal male genomic DNA. Results: aCGH profiles for pools of 10 cells using both WGA methods were very similar to unamplified SKBR3 genomic DNA. Signal to noise (S:N) was significantly higher with the REPLI-g amplified DNA compared to PicoPLEX, but single cells did not show similar profiles of genomic aberrations to unamplified genomic DNA until at least 3 cells were pooled before amplification. In contrast, single cells amplified using PicoPLEX demonstrated genomic aberrations consistent with unamplified genomic DNA. Fixation did not appear to adversely affect PicoPLEX WGA performance, but DNA from fixed cells could not be amplified with REPLI-g. Conclusions: Diagnostic decisions can potentially be made from molecular data derived from small numbers of CTCs requiring WGA methods that provide accurate results in downstream analysis. WGA methods should be selected based upon the molecular data required. In this study, higher S:N indicate that REPLI-g may be more sensitive for finding smaller genomic changes than PicoPLEX, however PicoPLEX generates more consistent data with single fixed cells. Citation Format: Rachel H.V. Needham, Arturo B. Ramirez, Iman Kishawi, Jackie L. Stilwell, Eric P. Kaldjian. Comparison of whole genome amplification methods on single and pooled cells for comparative genomic hybridization array analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3615.

Abstract P2-02-11: Detection of activating estrogen receptor 1 (ESR1) mutation on single circulating tumor cells from metastatic breast cancer patients

Abstract Background: 65% of primary breast cancers express the estrogen receptor α (ERα) and the mainstay of treatment are therapies that result in selective estrogen receptor modulation (SERM) of estrogen deprivation (aromatase inhibitors, AIs). Even thought endocrine therapy resulted in reduced recurrence and mortality, a significant portion of patients relapse with a metastatic disease and subsequently progress while of therapy for advanced disease (endocrine resistance). Recent evidence showed that activating hot spot mutation in the ligand binding domain of the ERα are acquired on treatment (frequency of 20%) and can drive resistance to endocrine therapy. Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material and the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies. These features suggest that the detection of ESR1 mutation on single CTC may be a useful biomarker for therapy guidance. Purpose: Investigate the incidence and heterogeneity of ESR1 mutational status within single CTCs isolated from individual metastatic breast cancer patients (mBCs), combining the FDA approved CellSearch® system for enumeration of CTCs with the DEPArrayTM technologies. Methods: CTCs were enriched and enumerate by CellSearch® in 7.5 ml blood samples collected from 21 mBCs according to standard protocol. Each CTC-enriched sample with at least 20 CTCs was recovered from Veridex cartridge and loaded into the DEPArrayTM A300K chip, since the DEPArrayTM analyzed only the 66% of the sample volume loaded, according to the manufacturer's instructions. The chip scanning was performed by automated fluorescence microscope. The loaded cells were recovered as single cell and subdivided in tree different group: Cytokeratin (CK) positive ( Dapi+, CK+, ER-, CD45-); ER positive (Dapi+, ER+, CK+, CD45-); White Blood cells (WBCs) (Dapi+, CD45+, CK-, ER-). Single CTCs and WBCs were then submitted to whole genome amplification (WGA) using the Single Cell WGA kit (Yikon Genomics) according the manufacturer's instructions. Detection of target 14 ESR1 hot spot mutations was performed on ABI PRISM® 3700 genetic analyzer by target Sanger sequencing. Results: 3 out of 21 mBCs with ≥20 CTCs were sorted and a total of 65 cells were recovered. WGA and ESR1 mutational status were performed on a total of 25 cells (respectively 11 ER+, 6 CK+ and 8 WBCs). In 1 of the 3 patients, that failed 2 lines of chemotherapy and previous single agent endocrine therapy, molecular heterogeneity was detected among its ER+ cells. 4 of 5 ER+ cells were heterozygote for the Y537S while one cell was homozygous, maybe due to a loss of heterozygosity. Y537S is one of the most common mutations that leads to a ligand independent ER transcriptional activity that does not respond to endocrine manipulation. No mutations were reported in all the CK+ and WBC cells analyzed. Conclusions: This study demonstrates the feasibility of a non-invasive approach based on liquid biopsy in mBCs. Evaluation of ER status and early identification of ESR1 mutation in ER+ CTCs might allow to predict effect of the endocrine therapies and switching to other treatments before the emergence of metastatic disease. Citation Format: Paolillo C, Mu Z, Austin L, Nguyen T, Capoluongo E, Fortina P, Cristofanilli M. Detection of activating estrogen receptor 1 (ESR1) mutation on single circulating tumor cells from metastatic breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-11.

Abstract 1606: Single cell isolation and DNA analysis from circulating tumor cells using self-sorting nanowell plates

Abstract Introduction The heterogeneity of tumor cells dictates the need for analysis at the single cell level. In addition, gene expression can be altered during the course of the disease and is accelerated under the influence of therapy. This imposes the need for a tumor biopsy each time therapeutic intervention is required. Circulating Tumor Cells (CTC) represent a unique opportunity for a “real time liquid biopsy”. Current available CTC counting technologies are hampered by inefficiency to isolate individual CTC for further molecular characterization to unveil the best treatment strategy. Here we introduce a simple solution to obtain and analyze the genetic make-up of individual CTC from a CTC enriched cell suspension. Methods Healthy donor blood is drawn into CellSave tubes and spiked with cells of the prostate cancer cell line LNCaP. Samples are processed between 24 and 96 hours using the CellSearch system. The full content of the CellSearch cartridge is transferred to a self-sorting nanowell plate. After identification on a fluorescence microscope single and multiple LNCaP's and WBC are punched out of the plate using a needle and deposited into tubes by using an automated X,Y stage. The DNA is amplified using the Ampli-1 kit from Silicon Biosystems (SB). Whole Genome Amplification (WGA) products are labeled with Cy-3 and Cy-5 and hybridized to an Agilent sureprint microarray slide. Data was analyzed with the Agilent Genomic workbench software. To show the compatibility with different WGA kits, Self Sorting Nanowell plates with mixed and sorted, differentially stained LNCaP, PC-3 and SKBR3 cells were prepared. Single cells were punched into wells and amplified with three WGA kits: New England Biolabs (NEB) Single Cell WGA kit, the GE Genomiphi kit and the SB Ampli-1 Kit. Evagreen was added to follow the reactions in realtime. Specificity of the system was shown by Sanger sequencing of each cell line for two specific mutations in the ROBO-1 and Pten genes. Results Cells that are captured, stained and quantified by the CellSearch system have been isolated using self-sorting nanowell Plates. Array CGH using the Ampli-1 WGA products of these cells shows the identification of the LNCaP cell line for amplified single cell and multi cell samples. Whole Genome Amplified DNA was found to be produced in 60% of the wells for the NEB kit, 77% for the GE kit and 67% for the SB kit. The punched single cells are correctly identified by Sanger sequencing and detection of the mutations in the Robo-1 and the PTEN genes. Conclusions We introduced a self-sorting nanowell plate in combination with an efficient method for the isolation of single cells from a CTC enriched cell suspension. 68% of the single isolated cells can be amplified with different WGA kits and cells isolated with CellSearch can subsequently be used for downstream applications such as sequencing and arrayCGH Citation Format: Joost F. Swennenhuis, Arjan GJ Tibbe, Michiel Stevens, Nikolas H. Stoecklein, Rui Neves, Hien D. Tong, Cees J.M. van Rijn, Leon W.M.M Terstappen. Single cell isolation and DNA analysis from circulating tumor cells using self-sorting nanowell plates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1606. doi:10.1158/1538-7445.AM2015-1606