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Rapid flow-sorting to simultaneously resolve multiplex massively parallel sequencing products
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.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-4313-1601
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.

Place, publisher, year, edition, pages
2011. Vol. 1, no 108
Keyword [en]
Development, Molecular sequence data, Genomics, Methods
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-45658DOI: 10.1038/srep00108ISI: 000296055500001Scopus ID: 2-s2.0-84859768054OAI: oai:DiVA.org:kth-45658DiVA: diva2:452752
Funder
Knut and Alice Wallenberg Foundation
Note
QC 20111031Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2011-11-16Bibliographically 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)
Opponent
Supervisors
Note
QC 20111031Available from: 2011-10-31 Created: 2011-10-31 Last updated: 2011-11-01Bibliographically approved

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