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Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing
KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0003-4313-1601
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.

Place, publisher, year, edition, pages
2010. Vol. 11, no 140
Keyword [en]
sequencing, emPCR, FACS
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-24357DOI: 10.1186/1471-2164-11-140ISI: 000275835300001Scopus ID: 2-s2.0-77949406720OAI: oai:DiVA.org:kth-24357DiVA: diva2:347895
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
In thesis
1. Tagging systems for sequencing large cohorts
Open this publication in new window or tab >>Tagging systems for sequencing large cohorts
2010 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets.

This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized.

The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set.

In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained.

Place, publisher, year, edition, pages
Stockholm, 2010. 38 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:15
Keyword
next generation sequencing, genotyping, massive parallel sequencing, 454, Pyrosequencing, amplicon sequencing, enrichment, DNA barcodes
National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-24365 (URN)978-91-7415-706-2 (ISBN)
Presentation
2010-09-24, FA32, Roslagstullsbacken 21, Stockholm, AlbaNova, 10:15 (Swedish)
Supervisors
Note
QC20100907Available from: 2010-09-07 Created: 2010-09-06 Last updated: 2012-03-23Bibliographically approved
2. 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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