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Massively parallel analysis of cells and nucleic acids
KTH, School of Biotechnology (BIO), Gene Technology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Recent proceedings in biotechnology have enabled completely new avenues in life science research to be explored. By allowing increased parallelization an ever-increasing complexity of cell samples or experiments can be investigated in shorter time and at a lower cost. This facilitates for example large-scale efforts to study cell heterogeneity at the single cell level, by analyzing cells in parallel that also can include global genomic analyses. The work presented in this thesis focuses on massively parallel analysis of cells or nucleic acid samples, demonstrating technology developments in the field as well as use of the technology in life sciences.

In stem cell research issues such as cell morphology, cell differentiation and effects of reprogramming factors are frequently studied, and to obtain information on cell heterogeneity these experiments are preferably carried out on single cells. In paper I we used a high-density microwell device in silicon and glass for culturing and screening of stem cells. Maintained pluripotency in stem cells from human and mouse was demonstrated in a screening assay by antibody staining and the chip was furthermore used for studying neural differentiation. The chip format allows for low sample volumes and rapid high-throughput analysis of single cells, and is compatible with Fluorescence Activated Cell Sorting (FACS) for precise cell selection.

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences by constantly producing increasing amounts of data from one sequencing run. However, the reagent costs and labor requirements in current massively parallel sequencing protocols are still substantial. In paper II-IV we have focused on flow-sorting techniques for improved sample preparation in bead-based massive sequencing platforms, with the aim of increasing the amount of quality data output, as demonstrated on the Roche/454 platform. In paper II we demonstrate a rapid alternative to the existing shotgun sample titration protocol and also use flow-sorting to enrich for beads that carry amplified template DNA after emulsion PCR, thus obtaining pure samples and with no downstream sacrifice of DNA sequencing quality. This should be seen in comparison to the standard 454-enrichment protocol, which gives rise to varying degrees of sample purity, thus affecting the sequence data output of the sequencing run. Massively parallel sequencing is also useful for deep sequencing of specific PCR-amplified targets in parallel. However, unspecific product formation is a common problem in amplicon sequencing and since these shorter products may be difficult to fully remove by standard procedures such as gel purification, and their presence inevitably reduces the number of target sequence reads that can be obtained in each sequencing run. In paper III a gene-specific fluorescent probe was used for target-specific FACS enrichment to specifically enrich for beads with an amplified target gene on the surface. Through this procedure a nearly three-fold increase in fraction of informative sequences was obtained and with no sequence bias introduced. Barcode labeling of different DNA libraries prior to pooling and emulsion PCR is standard procedure to maximize the number of experiments that can be run in one sequencing lane, while also decreasing the impact of technical noise. However, variation between libraries in quality and GC content affects amplification efficiency, which may result in biased fractions of the different libraries in the sequencing data. In paper IV barcode specific labeling and flow-sorting for normalization of beads with different barcodes on the surface was used in order to weigh the proportion of data obtained from different samples, while also removing mixed beads, and beads with no or poorly amplified product on the surface, hence also resulting in an increased sequence quality.

In paper V, cell heterogeneity within a human being is being investigated by low-coverage whole genome sequencing of single cell material. By focusing on the most variable portion of the human genome, polyguanine nucleotide repeat regions, variability between different cells is investigated and highly variable polyguanine repeat loci are identified. By selectively amplifying and sequencing polyguanine nucleotide repeats from single cells for which the phylogenetic relationship is known, we demonstrate that massively parallel sequencing can be used to study cell-cell variation in length of these repeats, based on which a phylogenetic tree can be drawn.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2011. , xi, 71 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:22
Keyword [en]
Massively parallel sequencing, 454, Illumina, multiplex amplification, whole genome amplification, single cell, polyguanine, flow-cytometry
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-45671ISBN: 978-91-7501-123-3 (print)OAI: oai:DiVA.org:kth-45671DiVA: diva2:452821
Public defence
2011-11-18, Lennart Nilsson-salen, Nobels väg 15A, Karolinska Institutet, Solna, 10:00 (English)
Opponent
Supervisors
Note
QC 20111031Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2011-11-01Bibliographically approved
List of papers
1. High-Density Microwell Chip for Culture and Analysis of Stem Cells
Open this publication in new window or tab >>High-Density Microwell Chip for Culture and Analysis of Stem Cells
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2009 (English)In: PLos ONE, ISSN 1932-6203, Vol. 4, no 9, e6997- p.Article in journal (Refereed) Published
Abstract [en]

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.

Keyword
animal cell; article; biochip; cell cloning; cell culture; cell differentiation; cell screening; cell selection; controlled study; embryo; embryonic stem cell; female; flow cytometry; human; human cell; microwell chip; molecular imaging; mouse; neural stem cell; nonhuman; stem cell; animal; C57BL mouse; cell separation; culture technique; cytology; DNA microarray; equipment design; metabolism; methodology; nerve cell
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11661 (URN)10.1371/journal.pone.0006997 (DOI)000269796300013 ()2-s2.0-70349206057 (Scopus ID)
Note
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2011-10-31Bibliographically approved
2. Flow cytometry for enrichment and titration in massively parallel DNA sequencing
Open this publication in new window or tab >>Flow cytometry for enrichment and titration in massively parallel DNA sequencing
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2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 8Article in journal (Refereed) Published
Abstract [en]

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.

Identifiers
urn:nbn:se:kth:diva-12402 (URN)10.1093/nar/gkp188 (DOI)000265953000007 ()2-s2.0-65849474143 (Scopus ID)
Note
QC 20100416Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2017-12-12Bibliographically approved
3. Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing
Open this publication in new window or tab >>Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing
2010 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, no 140Article in journal (Refereed) Published
Abstract [en]

Background

In addition to shotgun sequencing, next generation sequencing has been shown to be suitable for deep sequencing of many specific PCR-amplified target genes in parallel. However, unspecific product formation is a common problem in amplicon sequencing since these fragments are difficult to fully remove by gel purification, and their presence inevitably reduces the number of mappable sequence reads that can be obtained in each sequencing run.

Results

We have used a novel flow cytometric sorting approach to specifically enrich Roche/454 DNA Capture beads carrying target DNA sequences on their surface, and reject beads carrying unspecific sequences. This procedure gives a nearly three-fold increase in the fraction of informative sequences obtained. Presented results also show that there are no significant differences in the distribution or presence of different genotypes between a FACS-enriched sample and a standard-enriched control sample.

Conclusions

Target-specific FACS enrichment prior to Roche/454 sequencing provides a quick, inexpensive way of increasing the amount of high quality data obtained in a single sequencing run, without introducing any sequence bias.

Keyword
sequencing, emPCR, FACS
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-24357 (URN)10.1186/1471-2164-11-140 (DOI)000275835300001 ()2-s2.0-77949406720 (Scopus ID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note
QC 20100906Available from: 2010-09-06 Created: 2010-09-03 Last updated: 2017-12-12Bibliographically approved
4. Rapid flow-sorting to simultaneously resolve multiplex massively parallel sequencing products
Open this publication in new window or tab >>Rapid flow-sorting to simultaneously resolve multiplex massively parallel sequencing products
2011 (English)In: Scientific Reports, ISSN 2045-2322, Vol. 1, no 108Article in journal (Refereed) Published
Abstract [en]

Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent sequencing are based on amplification of library fragments on the surface of beads prior to sequencing. Commonly, libraries are barcoded and pooled, to maximise the sequence output of each sequence run. Here, we describe a novel approach for normalization of multiplex next generation sequencing libraries after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are prepared by fluorescent tagging of complementary sequences and then resolved by high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol is simple and provides an even sequence distribution of multiplex libraries when sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion PCR beads are removed, the approach gives rise to a substantial increase in sequence quality and mean read length, as compared to that obtained by standard enrichment protocols.

Keyword
Development, Molecular sequence data, Genomics, Methods
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-45658 (URN)10.1038/srep00108 (DOI)000296055500001 ()2-s2.0-84859768054 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation
Note
QC 20111031Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2011-11-16Bibliographically approved
5. Interrogation of polyguaninine nucleotide repeat variability in human T-cells by whole genome sequencing of single cells
Open this publication in new window or tab >>Interrogation of polyguaninine nucleotide repeat variability in human T-cells by whole genome sequencing of single cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Polyguanine nucleotide repeats exhibit a greater degree of variation than the average for the genome as a whole. This is partly due to polymerase slippage that causes insertions or deletions in these repeat sequence. The high variability of these repeats makes them useful for tracking the differentiation and fate of cells within tissues and organs. However, the same factors that create this variability also give rise to technical difficulties in DNA amplification and massively parallel DNA sequencing. In the study reported herein, we investigated shotgun sequence data from a standard multi-cell sample as well as sequence data for four single cells from the same individual. This was used to assess sequence quality in whole genome amplified single cell material and to investigate variability in homopolymeric regions between individual T-cells. In a more focused study, a selected set of polyG loci in single cells for which the phylogenetic relationship was known, were amplified and sequence determined. Based on the length differences in polyG repeats between the eight cells a phylogenetic tree was constructed, that was very similar to the known tree.

Keyword
DNA sequencing, whole genome amplification, single cell, whole genome sequencing, homopolymer, HiSeq2000, massively parallel sequencing, NGS
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-45659 (URN)
Note
QS 2011Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2011-10-31Bibliographically approved

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