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Library Preparation for High Throughput DNA Sequencing
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0002-2219-0197
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Order 3 billion base pairs of DNA in the correct order and you get the blueprint of a human, the genome. Before the introduction of massively parallel sequencing a little more than a decade ago it would cost around $10 million to get this blueprint. Since then, sequencing throughput and cost have plummeted and now that figure is around $1000, and large sequencing centres such as the National Genomics Infrastructure in Stockholm is sequencing the equivalent of 25 human genomes per hour. The papers that form the basis of this thesis cover different aspects of the rapidly expanding DNA sequencing field.

 

Paper I describes a model system that employ massively parallel sequencing to characterize the behaviour of type IIS restriction enzymes. Enzymes are biological macromolecules that catalyse chemical reactions in the cell. All commercially available sequencing systems use enzymes to prepare the nucleic acids before they are loaded on the machine. Thus, intimate knowledge of enzymes is vital not only when designing new sequencing protocols, but also for understanding the limitations of current protocols. Paper II covers the automation of a library preparation protocol for spatially resolved transcriptome sequencing. Automation increases the sample throughput and also minimises the risk of human errors that can introduce technical noise in the data. In paper III, the power of massively parallel sequencing is employed to describe the RNA content of the endometrium at two different time points during the menstrual cycle. Finally, paper IV covers the sequencing of highly degraded nucleic acids from formalin fixed, paraffin embedded samples. These samples often have a rich clinical background, making them extremely valuable for researchers. However, it is challenging to sequence these samples and this study looks at the impact that different preparation kits have on the quality of the sequencing data. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , 56 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:1
Keyword [en]
DNA, RNA, sequencing, massively parallel sequencing, library preparation, automation, genome, transcriptome
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-196560ISBN: 978-91-7729-212-8OAI: oai:DiVA.org:kth-196560DiVA: diva2:1048978
Public defence
2017-01-13, Air & Fire, Science for Life Laboratory, Tomtebodavägen 23, Solna, 13:00 (English)
Opponent
Supervisors
Note

QC 20161124

Available from: 2016-11-24 Created: 2016-11-16 Last updated: 2016-11-24Bibliographically approved
List of papers
1. Endonuclease specificity and sequence dependence of Type IIS restriction enzymes
Open this publication in new window or tab >>Endonuclease specificity and sequence dependence of Type IIS restriction enzymes
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 1, e0117059Article in journal (Refereed) Published
Abstract [en]

Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

Place, publisher, year, edition, pages
Public Library of Science, 2015
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-105132 (URN)10.1371/journal.pone.0117059 (DOI)000348732100060 ()2-s2.0-84922424353 (ScopusID)
Funder
EU, FP7, Seventh Framework Programme, 222913Swedish Foundation for Strategic Research
Note

Updated from Submitted to Published. QC 20150407

Available from: 2012-11-16 Created: 2012-11-16 Last updated: 2016-11-22Bibliographically approved
2. An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries.
Open this publication in new window or tab >>An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries.
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 37137Article in journal (Refereed) Published
Abstract [en]

Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context. In this study, a protocol for retaining spatial information of transcripts has been adapted to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-196766 (URN)10.1038/srep37137 (DOI)000388080900001 ()27849009 (PubMedID)2-s2.0-84995665687 (ScopusID)
Funder
Knut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research Swedish Research Council
Note

QC 20161124

Available from: 2016-11-21 Created: 2016-11-21 Last updated: 2016-12-14Bibliographically approved
3. Comprehensive RNA sequencing of healthy human endometrium at two time points of the menstrual cycle
Open this publication in new window or tab >>Comprehensive RNA sequencing of healthy human endometrium at two time points of the menstrual cycle
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2016 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Endometrial receptivity is crucial for implantation and establishment of a normal pregnancy. The shift from proliferative to receptive endometrium is still far from understood. In this paper we comprehensively present the transcriptome of the human endometrium by comparing endometrial biopsies from proliferative phase with consecutive biopsies 7-9 days after ovulation. The results show a clear difference in expression between the two time points using both total and small RNA sequencing.  3297 mRNAs, 516 long non-coding RNAs and 102 small non-coding RNAs were identified as statistically differentially expressed between the two time points. We show a thorough description of the change in mRNA between the two time points and display lncRNAs, snoRNAs and snRNAs not previously reported in the healthy human endometrium. In conclusion this paper reports in detail the shift in RNA expression from the proliferative to receptive endometrium.

Keyword
RNA-seq, endometrium, menstruation cycle, differential expression, small RNA, total RNA
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:kth:diva-184156 (URN)
Funder
Swedish Research Council
Note

QC 20161124

Available from: 2016-03-29 Created: 2016-03-29 Last updated: 2016-11-24Bibliographically approved
4. Evaluation of methods for whole genome and transcriptome sequencing from nanograms of FFPE samples
Open this publication in new window or tab >>Evaluation of methods for whole genome and transcriptome sequencing from nanograms of FFPE samples
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The most widely used method for the preservation of clinical tissue specimens is formalin fixation and paraffin embedding (FFPE). Simultaneous analysis of RNA and DNA from samples preserved using this method have long proved problematic, primarily due to lack of material. Here, we describe an attempt to build a complete analysis package for RNA and DNA extracted from single tissue sections. The workflow includes quality control of the extracted material, library preparation and data analysis. We extract DNA with varying integrity from FFPE sections and subject them to whole genome sequencing using two library preparation methods, Illumina TruSeq Nano using the Illumina NeoPrep and Rubicon Genomics ThruPlex. We are able to obtain some usable data, albeit with high duplication rates, demonstrating both the possibilities and challenges of sequencing damaged DNA. Two different approaches to transcriptome sequencing are assessed, the TruSeq RNA Access library preparation kit from Illumina and the SMARTer Stranded Total RNA-Seq Kit - Pico Input from Clonetech. The sequence capture approach of the TruSeq kit is shown to be more robust to low integrity RNA compared to the SMARTer kit. However, the SMARTer kit needs much less starting material and is able to yield data about all transcripts, not just protein coding mRNA.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-196775 (URN)
Note

QC 20161124

Available from: 2016-11-21 Created: 2016-11-21 Last updated: 2016-11-24Bibliographically approved

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