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Interrogation of polyguaninine nucleotide repeat variability in human T-cells by whole genome sequencing of single cells
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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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 [en]
DNA sequencing, whole genome amplification, single cell, whole genome sequencing, homopolymer, HiSeq2000, massively parallel sequencing, NGS
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-45659OAI: oai:DiVA.org:kth-45659DiVA: diva2:452769
Note
QS 2011Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2011-10-31Bibliographically approved
In thesis
1. Massively parallel analysis of cells and nucleic acids
Open this publication in new window or tab >>Massively parallel analysis of cells and nucleic acids
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
Massively parallel sequencing, 454, Illumina, multiplex amplification, whole genome amplification, single cell, polyguanine, flow-cytometry
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-45671 (URN)978-91-7501-123-3 (ISBN)
Public defence
2011-11-18, Lennart Nilsson-salen, Nobels väg 15A, Karolinska Institutet, Solna, 10:00 (English)
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Note
QC 20111031Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2011-11-01Bibliographically approved

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