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Practical training Molecular Biotechnology
WS 2016/2017
AmpliSeq RNA Sequencing
Relative quantitation of cell stress specific gene expression using AmpliSeq RNA sequencing in human keratinocyte after plasma treatment.
Relative Quantifizierung von Zellstress-spezifischen Genen mittels AmpliSeq RNA Sequenzierung in menschlichen Keratinozyten nach Plasma-Exposition.
Script:
Dr. rer. nat. G. Chometon-Luthe
Module: Molecular Biotechnology, Prof. Dr. K. Hennes
Master course of studies: Bio- und Nanotechnology
South Westphalia of University of Applied Sciences
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Content
Allgemeines zum Praktikum ... 4
Allgemeine Hinweise zur Organisation ... 4
Allgemeine Regeln für das Praktikum ... 4
Sicherheitsvorschriften ... 4
Chapter I ... 5
1. Introduction to sequencing ... 5
Chapter II ... 8
2. NGS: Ion Torrent Personal Genome Machine ... 8
2.1. The Chip ... 8
2.2. Application of sequencing with the PGM machine ... 9
2.3 Workflow ... 9
2.4. Step 1: Template preparation ... 10
2.4.1. Preparation of a DNA template and creation of a library ... 11
Designed primer panel ... 12
2.4.2. Preparation of a sequencing matrix ... 13
2.5. Step 2: Sequencing: ... 16
2.6. Step 3: Sequence alignment and assembly ... 16
Chapter III ... 18
3. The tools of genetic engineering ... 18
3.1. Good Laboratory Practice ... 18
3.2. Control samples and normalization ... 19
3.3. Use of Quality Control ... 19
4. Preliminary work: ... 20
4.1. HaCaT Cell line and treatment ... 20
4.2. Isolation total RNA (Pure Link RNA Mini Kit, Life Technologies) ... 21
5. Preparation of the library (Ion AmpliseqTM RNA Library Kit, Life Technologies) ... 22
5.1. Workflow ... 22
5.2. Required materials and equipment... 22
5.3. Procedure ... 23
5.3.1. Reverse Transcription PCR ... 23
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5.3.2. Amplify targets: ... 24
5.3.3. Partially digest primer sequences ... 24
5.3.4. Ligate adaptors to the amplicons and purify... 24
5.3.5. Purify the unamplified library: ... 25
5.3.5. Amplify the library and purify ... 26
5.3.6. Quantify the library and dilute for the template preparation (Qubit dsDNA HS Assay Kit) 27 5.3.5. Combine barcoded libraries from different samples ... 27
6. Preparation of the sequencing template (Ion PGM Template OT2 200 Kit) ... 28
6.1. Workflow ... 28
6.2. Required materials and equipment... 28
6.3. Procedure ... 30
6.3.1. Ion OneTouch™ 2 Instrument ... 30
6.3.2. Enrich template-positive OT2 200 Ion Sphere™ Particles with Ion OneTouch™ ES ... 32
7. Sequencing (Ion PGM Sequencing 200 Kit v2, Life Technologies) ... 34
7.1. Workflow ... 34
7.2. Required materials and equipment... 34
7.3. Procedure ... 35
7.3.1. Initialize the Ion PGM™ System (about 1 hour) ... 35
7.3.2. Sequencing ... 36
8. Check Run Report and insight into Data Analysis... 38
8.1. Check Run Report ... 38
8.2. Insight into Data Analysis ... 39
8.2. Creation of posters and poster presentation ... 39
Glossary (english-deutsch) ... 42
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Allgemeines zum Praktikum
Bei Versuchsantritt wird von jedem Praktikanten erwartet, dass er sich über den geplanten Versuchsablauf und die geplanten Experimente informiert hat und sehr gut vorbereitet hat.
Allgemeine Hinweise zur Organisation o 4 Praktikumstage à 5,5 Stunden.
o Die Versuche werden in den Räumen P001 und H613 durchgeführt.
o Vor Versuchsbeginn bitte Jacken, Mäntel, Taschen etc. in Raum P005 ablegen.
o Achtung: In Raum P005 dürfen keine Kittel gelagert werden!
o Eigene Laborkittel ist mitzubringen.
Allgemeine Regeln für das Praktikum
o Innerhalb des Praktikums ist ein halber Fehltag gestattet; ein ärztliches Attest ist erforderlich.
Bei mehr als einem halben Fehltag gilt das Praktikum als nicht bestanden.
o Bei einer Verspätung von mehr als 30 Minuten gilt dieser Tag als Fehltag.
o Während des Praktikums gilt im gesamten Praktikumsbereich (Räume P001 und P613) ein absolutes Mobiltelefonen und MP3-Playern Verbot.
o Orientieren Sie sich im Labor und an Ihrem Arbeitsplatz. Kontrollieren Sie, ob alle nötigen Gerätschaften vorhanden und funktionsfähig sind.
o Halten Sie Ihren Arbeitsplatz bitte stets sauber und ordentlich! Papierreste, Tupfer, Watte o.
ä. landen nach Gebrauch im Papierkorb. Glasbruch, Objektträger und Deckgläser sind im Sonderbehälter für Glasabfall zu entsorgen.
o Schäden werden auf Kosten des Verursachers behoben.
Sicherheitsvorschriften
o Sämtliche Vorsichtsmaßnahmen für das Arbeiten in biotechnologischen Laboratorien, die in den Richtlinien für Laboratorien (TRBA 100 und GUV19.17, Ausgabe November 1998) dargelegt sind, sind einzuhalten.
o Das Tragen von Kittel und Schutzbrille ist im gesamten Laborbereich Pflicht.
o Zellkulturreste und -überstände dürfen nicht unbehandelt in die Ausgüsse oder in den Hausmüll gegeben werden. Sie müssen unbedingt vor der Entsorgung autoklaviert werden.
o Verschüttetes biologisches Material muss sofort mit Desinfektionsmitteln (z.B. 70 %igem Ethanol) behandelt und mit Papiertüchern aufgewischt werden.
o Essen, Trinken und Rauchen ist im Laborbereich verboten, ebenso das Lagern von Lebensmitteln.
o Bei Arbeiten mit Gefahrstoffen aller Art sind die entsprechende Betriebsanweisung und das Sicherheitsdatenblatt zu beachten. Vor Aufnahme der Arbeit mit einem Gefahrstoff ist das Sicherheitsdatenblatt (MSDS) zu lesen. Lesen Sie die MSDS der benötigten Produkte! Beachten Sie die Besonderheiten bezüglich der Entsorgung der Chemikalien.
o Die Standorte von Feuerlöschern, Verbandkästen, Notduschen, Augenduschen und die Notausgänge müssen bekannt sein.
o Spitzes oder scharfkantiges Material muss in Kunststoffbehältern so gesammelt werden, dass eine Verletzung von Mitarbeitern ausgeschlossen ist.
o Geräte dürfen ohne Anweisung nicht benutzt werden.
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Chapter I
1. Introduction to sequencing
So far, four generations of sequencing technology expand the field of biological science.
The Sanger method is considered as the first generation technology. This technology is based on the chain termination method or Sanger dideoxy sequencing (Sanger et al. 1977). In the modern Sanger sequencing, the DNA sequencing is simplified by the use of fluorescently labeled ddNTPs or fluorescently labeled primers instead of radioactive material. These platforms are still commonly used.
The second generation of sequencing is called the “NGS” or Next Generation Sequencing. Roche, Illumina and Life Technologies/ Thermo Fischer Scientific are the main NGS platform providers. The technique is characterized by a massively parallel sequencing. For most of the NGS platforms, the template preparation is a multistep process and consists of fragmentation of DNA, addition of adapters, immobilization of the templates to a solid support and finally clonal DNA amplification (Fuller et al.
2009).
The third and the fourth generation technology, in contrast, do not require any amplification of DNA fragments prior to sequencing as single DNA or RNA molecules can be sequenced in real time (Schadt et al. 2010). The sequencing strategies are either direct imaging of DNA using microscopy techniques (i.e. electron microscopy), sequencing DNA molecules passing a nanopore (Oxford Nanopore) or direct observation of DNA synthesis (Pacific Biosciences).
For a detailed description of the currently available sequencers, the reader is referred to selected reviews (Mardis 2008; Metzker 2010; Ku und Roukos 2013; Fuller et al. 2009).
Sequencing is not only the process of determining the precise order of nucleotides within a DNA molecule; it is also a technology which aims to understand how the genetic code governs cellular function. This technology finds its applications in all fields involving genome and transcriptome research (Figure 1). According to prior knowledge of the sequencing region or not, a distinction must be made between re-sequencing and de-novo sequencing.
Genome sequencing: One obvious application of a sequencer is the sequencing of genome. From 1990 to 2003, the realization of the human genome project was a milestone in biomedical research (Human Genome Project Information Archive, HGMIS, (Lander et al. 2001). To date, in accordance to NCBI and GOLD, over 1000 new genomes (de novo) are sequenced per year.
Microbial sequencing:One application of microbial sequencing is a metagenomic study of a bacterial or viral community. Metagenomic refers to a collection of genes issues from a corpus of microbial species found the environment. The aim of a metagenomic study is the discovery and characterization of microbial organisms without prior knowledge of the sequencing region or genome (de novo sequencing). Sometimes it is enough to sequenced only a targeted region of the bacterial 16S rRNA (Whiteley et al. 2012; Junemann et al. 2012).
Another important application of microbial sequencing is the typing of individual strains of bacteria or viruses to monitor or characterize them in context of a high contagious disease outbreak. In early 2011, there has been an unusual outbreak of multidrug-resistant EHEC enterohemorrhagic E. Coli O104:H4 in Germany (Mellmann et al. 2011). Thanks rapid NGS sequencing, the whole genome of the virulent strains was characterized and the source of the resistance analyzed. These kinds of study are usefully to better monitor prevention and therapy in a future outbreak.
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Figure 1: Upper figure: The total DNA content in a cell is referred to as the genome. The exons are mRNA- encoding entities. This part of the genome represents the exome. The total content of RNA in a cell at a given time point is referred to as the transcriptome. The entire set of proteins expressed represents the proteome (inspired from {Robinson et al 2004}. Lower figure: Application of Next-Generation-Sequencing techniques (inspired from {Nowrousian et al, 2010).
Genome organization: 3D structures between DNA and protein within the nucleus can also be analyzed by sequencing using the methods of Hi-C, ChIP-Seq or Methyl-Seq (Figure 1). Hi-C is a technique which enables to explore the 3D-chromatin structure based on Chromosome Conformation Capture (Belton et al. 2012). ChIP-Seq (chromatin immunoprecipitation)-Seq and Methyl-Seq permit to study the interaction between nucleosomes, transcription factor binding sites and methylation states of chromatin proteins. These methods are all used with a reference genome (re-sequencing).
Targeted DNA or RNA Sequencing: In the study of diseases (i.e. cancer, genetic disorder) and for the development of drugs is the identification of biomarkers a crucial step. A biomarker can be found
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among DNA regions (Exome or not), different RNA molecules (coding RNA or non-coding ncRNA), or in form of a protein among the Proteome (Dancey et al. 2012). So, from genome to transcriptome, both the analysis of gene variation (DNA level) and the analysis of the expression pattern (RNA and protein level) are important (are those genes expressed and what affects their expression?).
For this reason, targeted DNA sequencing has become a powerful methodology to identify and study specific gene variants (hotspot mutations in form of Single Nucleotide Polymorphism (SNPs), Insertion and Deletion (Indels), large deletions). For example, the screening of several lung cancer patients DNA by NGS has accelerated the finding of driver mutations, leading to the rapid development of targeted agents (Scarpa et al. 2013).
Targeted RNA sequencing is an alternative method to gene microarray or Taqman assay to focus on selected sets of protein-coding genes or genomic regions. Targeted RNA sequencing aims to analyze the expression profile of specific genes: the application goes to gene regulation analysis, correlation of gene expression with phenotypic information to cellular pathway analysis.
The advantage of RNA targeted sequencing vis-à-vis gene microarray and Taqman assay is the low amount of RNA that is needed for sequencing, the realization of allele-specific expression analysis and the detection of fusion transcripts. Therefore, this method might be better suited to target biomarkers in diagnostic testing.
This methodology is not yet mature. Few publications using this assay can be found under “targeted RNA sequencing” under PubMed, the first publication appearing in 2012 (Mercer et al. 2012; Blomquist et al. 2013; Hutchinson et al. 2013; Poole et al. 2014; Mercer et al. 2014; Fu et al. 2014). The method is still ongoing improvement in the field of template preparation, sequencing platforms and analysis of the data (field of bioinformatics).1
1 The definition of some selected words highlighted in bold can be found in the glossary at the end of the script.
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Chapter II
2. NGS: Ion Torrent Personal Genome Machine
The sequencing platform used here is the Ion PGMTM (Personal Genome Machine) by Life Technologies (Thermo Fischer Scientific Inc.), a so-called benchtop sequencer (Rothberg et al. 2011). This NGS- platform was invented by Ion Torrent, a business unit of Life Technologies, and launched end of December 2010 by Life Technologies. The particularity of this sequencer is the semiconductor chip, its speed (less than two hours sequencing time) and the (relative!) low cost of the sequencing process which enables small labs to get in touch with this technology.
2.1. The Chip
Ion Torrent sequencing technology is confined in a semiconductor chip (Figure 2). There are three different chips available: Ion 314TM chip, Ion 316TM chip and Ion 318TM chip, each chip containing an increasing number of micro-wells (1, 3 million micro wells, 6 million sensors and 12 million sensors respectively). This corresponds to a recording of 100 000 to 8 million reads.
Figure 2: The Ion chip is the machine. At the center are 1, 4 million wells. Each micro-well contains an Ion SphereTM particle coupled to DNA template. After incorporation of a nucleotide, a proton is released and the pH changes. Underneath, the sensing plate senses pH variation and translate it into voltage variation and lately into digital information (from Life Technologies and Rothberg et al. 2011).
The chip is “simply” translating pH variation into digital information (Figure 2). The local change of pH originates from the release of a proton when a new nucleotide is incorporated by the polymerase in the DNA molecule. In the PGM machine, the chip is consecutively flooded with one nucleotide after another (dATP, dTTP, dGTP, dCTP). If a nucleotide complements the sequence of the DNA molecule in a particular micro-well, it will be incorporated and hydrogen ions are released (Figure 3). Are there successively several identical nucleotide bases on the DNA strand, the voltage increases accordingly and the chip records multiple identical bases. If the nucleotide does not match with the DNA sequence, then no voltage change is recorded.
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Figure 3: Sequencing by synthesis: based upon the DNA template, the PGM platform allows the synthesis of a new strand. Thereby, the DNA template can be sequenced. A proton is released when a nucleotide is incorporated by the polymerase in the DNA template (from Life Technologies application note).
2.2. Application of sequencing with the PGM machine
We will use the smallest chip Ion314TM with its capacity to record about 100 000 reads and about 40 Mb data. Because of this small scalability, the applications are limited to (See Figure 1):
small genome sequencing (microbial genome)
targeted DNA sequencing
targeted RNA sequencing
We will use the Ion AmpliSeqTM technology (targeted RNA sequencing) to analyze the expression of selected genes which play a crucial role in cell stress response.
2.3 Workflow
For most of the NGS sequencing platforms, the whole process of sequencing is sub-divided in three main operational processes:
Step 1: Template preparation
Step 2: Sequencing
Step 3: Sequence alignment and assembly
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Which approach should be used to sequence a sample depends on several factors:
o the chosen application here, targeted RNA sequencing o the length of the target here, max. 200 bp
o the complexity of the sample here, isolated cells from culture
o the number of the samples here, 4
o the prior knowledge of the targeted Sequencing region (de novo sequen-
cing or resequencing) here, resequencing
The workflow for the PGM sequencing under our conditions is as follow (Figure 4):
Figure 4: Workflow of Ampliseq RNA sequencing
2.4. Step 1: Template preparation
The template preparation includes the handling of the DNA sample before the actual sequencing process (Figure 5).
The aim is:
o to get a DNA template with specific length because sequencer has defined average read length.
The read length is dependent of the capability of the sequencer machine to sequence short DNA sequences (so called reads) that range from 40 to 900 nucleotides in length. For ex. The Sanger sequencing average read length is 800 bp. Our PGM has an average read length of about 400 bp (An exception is the single-molecule real-time sequencing from Pacific Biosciences (NGS) with a max. read length of 30.000 bases.)
o to create a library: The aim of this step is the ligation of a couple of adapters P1 and barcode adapter A to the 3´and 5´region of specific chosen DNA sequences to be analyzed. A library is a collection of DNA sequences one is interested in.
o to clonally amplify the library and isolate the Ion Sphere Particles: With the help of these adapters, each amplicon can be later isolated and amplified before being sequenced in a microwell on the chip.
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Figure 5: Step 1: The template preparation consists of the conversion of RNA into cDNA, the amplification of specific region of cDNA using a primer panel, the ligation of adaptors to each amplicons and finally the amplification of the library.
2.4.1. Preparation of a DNA template and creation of a library
As we here are interested in analyzing the differential expression of certain genes, we will collect the RNA of our cells treated under different conditions. Total RNA is isolated using organic extraction methods (gold standard method) and spin silica column. The isolated RNA fraction contains all RNA molecules one cell can have.
It is much easier to work with DNA than with RNA. Therefore, the total isolated RNA will be transcript in cDNA by reverse transcription (RT) using random primers (Figure 5).
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Figure 6: Diagram of RT-PCR: total RNA is used as template for the cDNA synthesis. From these cDNA molecules, only specific targets are amplified by PCR
In a first step, the first strand cDNA synthesis is performed using total RNA as template, the reverse transcriptase SuperScript III, a high concentration of dNTP’s and random primers (hexamers).
SuperScript III is an enzyme that is able to synthesize first-strand cDNA from total RNA at a temperature range of 42-55°C. This enzyme has a reduced RNase H activity and increased thermal stability.
In a next step, a PCR reaction takes place with another polymerase, the Taq DNA Polymerase, its specific buffer, dNTPs and primers specific for the genes of interest (designed primer panel, see below).
Designed primer pairs hybridize up and down of specific region of transcripts of interest. At the end of the PCR procedure, the amplified regions are called amplicons. Each amplicon is about 150 bp long (Figure 7).
The whole procedure is called RT-PCR (here two-enzyme approach) (Nolan et al. 2006).
Designed primer panel
The Databank NCBI (National Center for Biotechnology Information) is a useful tool by providing access to biomedical and genomic information. Under Resource “Genes and Expression” or “Gene”, one can find the database of genes from genomes that have been completely sequenced. After an active research, a list of genes implicated in keratinocyte oxidative stress was established. The information data is based on published scientific research. From this list, 28 genes were selected based on their implications in different stress or survival pathways (Appendix Table 4). Only the protein-coding genes were selected.
28 PCR primers have been ordered via Ion Primer Tools from Life Technologies services. The primers are generated based on standard software tools like Primer3.
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A selected gene can have several transcripts, like for ex. TNFSF1. In this case, the primer pairs were designed to recognize all the transcripts.
The Library
After the amplification of the targets, the library is finally created as the Ion Torrent adaptors are ligated to each amplicons. Via a DNA ligase, the P1 adaptors and the barcode adaptors X are annealed to the 5´end of each amplicons (Figure 7). Barcodes are small nucleotide sequences which enable to mark each library and distinguish one from another when running multiple libraries on a single chip.
Figure 7: Adaptor sequence (P1 adaptor, Barcode and P1 adaptor) required to generate the amplicon sequencing library.
2.4.2. Preparation of a sequencing matrix
The aim of the preparation of a sequencing matrix is to:
to prepare Ion Sphere Particles (ISPs)
then amplify clonally the amplicons linked to a sphere particles (OneTouch2 machine)
And finally to enrich the ISPs-amplicon particles in the OneTouch ES machine before the sequencing step.
In the library solution, there are many amplicons (with adapters). All these amplicons need to be attached to special particles called Ion Sphere Particles (ISPs). The ISPs are microscopic beads with bound oligonucleotide (B) on their surface (Figure 8). In order to anneal the amplicons to the IPS, a special PCR will take place, called emulsion PCR in the presence of “ePCR primers” (Zhu et al. 2012).
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Figure 8: Schematic representation of how the amplicon libraries are annealed to the Ion Sphere Particles (ISPs):
the resulting templates can then be sequenced unidirectionaly (inspired from www.biorigami.com).
The emPCR-primer pairs used for the emulsion PCR consist of a B-trP1 PCR sequence complementary to the B sequence on the IPSs and complementary to the adaptors P1 sequence, and an ePCR-A-Biot sequence complementary to the adaptor A´ sequence. This second emPCR primer is a biotinylated primer i.e. it is labeled with a biotin protein.
The clonal amplification is done in the OneTouch 2 machine. This machine is a thermocycler with an integrated centrifuge. The emulsion PCR takes place in a “micro reactor” in a reaction filter which consists of an oil-water suspension. During the emPCR procedure, the B-P1_PCR primer anneals to P1 and the polymerase can extend the new strang. Then the complementary sequence is extended.
It is very important that one single amplicon sequence bind to one ISP (Ratio 1/1) and be amplified clonally in one micro reactor. To reach this optimal condition, the exact concentration of the library has to be determined, otherwise, several non-optimal conformations can occur (Figure 9).
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Figure 9: Schematic representation of the emulsion PCR which takes place in the micro reactors in the OneTouch2 machine (picture). Several conformations can occur.
Conformation 1: The micro reactor contains a sphere and different amplicons. After the PCR amplification, the ISP is polyclonal, i.e. the sphere is linked to different amplicons.
Conformation 2: The micro reactor contains a sphere and one amplicon. After the PCR amplification, the ISP is monoclonal, i.e. the sphere is linked to the same amplified amplicon.
Perfect!
Conformation 3: The micro reactor contains several spheres and one amplicon. After the PCR amplification, the ISP is monoclonal, but the amplification was suboptimal.
Conformation 4: The micro reactor contains a sphere, no amplicons. The PCR doesn´t take place.
Conformation 5: The micro reactor contains an amplicon, but no ISP is present.
The ion OneTouch ES is an enrichment system: it employs magnetic bead technology to concentrate the ISPs linked to DNA, so to maximize the sequencing yield.
The Ion PGM™ Enrichment Beads are streptavidin-coupled Dynabeads®. Dynabeads are super paramagnetic particles from uniformly spherical shape. Thanks their magnetic properties, the particles can be used to separate biological material when these are adsorbed or coupled to their surface.
Dynabeads MyOne Streptavidin C1 are coupled on their surface with streptavidin protein.
Magnetic Streptavidin beads are added to the ISPs binding to the biotinylated template from the emulsion PCR. Because biotin has a high affinity for streptavidin, the amplicons clonally amplified can be fished. The beads are immobilized with a magnet. A wash step removes the beads without DNA.
Denaturation with NaOH makes the DNA on the ISP single stranded and those particles are collected for sequencing.
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2.5. Step 2: Sequencing:
The sequencing procedure begins with the initialization of the sequencer: the system is prepared in detecting pH changes. Then, the chip is loaded with the templates (the amplicons attached to the ISPs) and the sequencing process is launched. It begins with the binding of Sequencing Primer to the A adaptor sequence. As the polymerase polymerizes the new strang, it reaches the Key sequence.
A “flow” is the event of exposing the chip to a particular dNTP (T, A, C, or G), followed by a washing step. The sequencing begins at the 3´of the Sequencing Primer, further on with the sensing of the Key Sequence “AGTC” (
Figure
11).Figure 10: Schematic representation of the templates. The sequencing process begins with the binding of the sequencing primers to the A adaptor sequence. Then the sequencing by synthesis takes place (here from right to left).
The sequence coverage per amplicons is registered as an ionogramme. It is the output of the signals in flow space and must be read “up-and-down” along with “left-to-right”. The height of the bar indicated how many nucleotides incorporate during the flow. Negative or zero flows indicate no nucleotide incorporation (Figure 11).
Figure 11: Ionogramme showing the number of flows and the respective nucleotide which was incorporated. One color codes for one nucleotide. The height of the bars corresponds to the quantity of one base encountered in a row.
2.6. Step 3: Sequence alignment and assembly
During a “run”, the data file .DAT integrates the raw data set (ionogramme). The data are then transferred from PGM to the Server. There, the raw signals are converted to Base Calls (A, T, C, G) and together with the quality data of the run (Phred Score) can be found and analyzed in the data file .FASTQ.
Before the alignment process begins, the reads are trimmed (elimination of adaptors sequences and portions of reads with bad quality), and filtered (elimination of small size reads and polyclonal reads).
17 Sequence alignment:
The NGS machines have the faculty to sequence at the same time millions of samples in a massive parallel sequencing process. This enables a higher throughput than the automated Sanger sequencing.
Most of the sequencers are not able to read long DNA template, the DNA sequence has to be shortened, either by cutting many times randomly or by amplify specific small target as here in targeted sequencing.
After sequencing, the reads has to be mapped back i.e. aligned against a reference genome (>3 billion nucleotides), i.e. a digital nucleic acid sequence database maintained and improved by the Genome Reference Consortium. Our reference genome is the human genome UCSC hg19 (University of California, Santa Cruz, human genome 19). The reference genome in NCBI is found under GRCh38 (Genome Reference Consortium Human Build 38) and is synonym of UCSC gh19.
Figure 12: Example of reads mapped to a reference sequence (top).
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Chapter III
3. The tools of genetic engineering
3.1. Good Laboratory Practice
In order to ensure reliable and contamination-free results, it is recommended to separate the “clean”
or pre-PCR area from the “dirty” or post-PCR area (Figure 13). The best layout is the two-room layout, one room for each area. However, if this configuration is not available but only one room, the separation of the two areas should be clearly labeled to reduce the potential of contamination.
Figure 13: General considerations for a unidirectional workflow.
PrePCR-sector (Clean): In this sector are located a micro-centrifuge, a set of pipettes and a thermal cycler.
The library set-up takes place within this area. A UV treatment minimizes contamination.
The isolation of total RNA requires a particular area with UV lamp. RNA is extremely susceptible to degradation and care must be taken to create a ribonuclease-free environment. RNases are everywhere!
Post-PCR-sector (Dirty): In this sector are located a set of pipettes, the Qubit, the PGM with server and Gas bottle, OneTouch 2 and ES. Here, the amplification mixture takes place, as well as the quantity control of the library following emPCR and the ion chip preparation.
Common GLP:
o Be aware to consequently move from “clean” to “dirty” area. Change the lab coat and separate sets of pipettes between both sectors.
o For all procedures use DNase/RNase-free consumables o Defrost all reagents on ice
o All reagents should be clearly labeled o Gloves should be often replaced.
o if possible, the reagents should be aliquot to minimize contamination (Primer-Solution…) o the laboratory bench should be clean with either a hypochlorite solution. When possible, use
UV-treatment.
o Generate master mixes if possible o Centrifuge reagents before opening.
o Open and close the reagent carefully.
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3.2. Control samples and normalization
All experiment is subject to variability along the different steps, caused by technical as well as biological variation. In order to identify relevant changes in gene expression, the data can be normalized using internal control gene (housekeeping genes) or external control (spikes-in) (Loven et al. 2012).
Here we will use two endogenous reference genes, TBP and MRPL13 (de Jonge, Hendrik J M et al. 2007;
Bar et al. 2009) for normalizing. The gene expression measurement is a relative quantification: in one sample, the expression level of a target is related to the reference genes, between different samples, one uses the same reference genes.
An ideal internal control gene should be uniformly expressed during all environmental system (no expression variation after treatment). Actually, there should be more than one reference gene and their expression pattern should be weak. Because the amplification control is intern to the sample, it co- purifies and co-amplifies with the targets. Also the different samples are processed simultaneously. All are subject of variability at the same degree.
For the negative amplification controls, no template control is used to check for non-specific signal arising from primer dimer or template contamination.
3.3. Use of Quality Control
The result of experiments like gene expression measurement by quantitative PCR, microarray, or sequencing where the quantification of amplification product is concern, depends highly on template quantity, quality and reliability (standardization) of the assay.
In order to minimize the variability, it is important to have quantity and quality control all long the assay (Figure 14).
Figure 14: Quantity and quality workflow during the process of Ampliseq RNA sequencing Here, we will proceed to quality control during several steps:
1) Starting material RNA: Assessment of RNA quality and quantity
Quantitation of RNA with UV Spectrophotometer measurement of A260.
Quality of RNA: control of its purity and control of its integrity 2) Quantify library: with Qubit DS HS kit
3) Quality control of ISPsa: with Qubit assay 4) Check run report after sequencing
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Chapter IV: Relative quantitation of cell stress specific genes using AmpliSeq RNA sequencing in human keratinocyte after plasma treatment .
4. Preliminary work:
“Plasma medicine” is an emerging field in the domain of biology and medicine. Non-thermal plasma (or non-equilibrium plasma) has proven itself as an excellent tool for treatment of cells or tissues.
It affects firstly the cell membrane and its permeabilization, so that it can be used for genetic transfection (Leduc et al. 2009). Another area of use is the sterilization of animal and human tissues or medical devices as it inactivates bacteria and spores (bactericidal effect) (Kong et al. 2009). In the domain of the surgery, plasma is used for blood coagulation (cauterization effect) without damaging the surrounding tissue. Furthermore, it can be used for the treatment of skin diseases like burn wound or cutaneous leishmaniasis as it stimulates wound healing and tissue regeneration. On the other side, application of plasma on mammalian cells can cause cell detachment, cell stress (Nitric Oxide release), necrosis or apoptosis, and therefore could be a promising tool in cancer treatment.
On the molecular level, however, not much is known about the cell responses after plasma treatments.
Here, using targeted RNA sequencing analysis, we aim to investigate the effect of plasma treatment on gene expression in keratinocytes in culture. We will profile 28 genes implicated in different pathways involved in cell stress and cell survival responses in untreated cells compared to plasma-treated cells.
4.1. HaCaT Cell line and treatment
Immortalized human keratinocytes HaCaT were grown in DMEM (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS), 0, 1 mg/ml gentamycin and 2, 5 µg/ml amphotericin at 37°C and 5% Co2 in a humidified atmosphere. HaCaT cells were plated in tissue culture dishes (9 cm2, TPP) in the respective medium. 24h later, as the cells reached sub confluence, the medium was replaced (500 µl) against fresh medium and the cells were either treated with plasma or let untreated.
Treatment of plasma consisted on a short exposure time of 37s at 25mm height, with a flow rate of 2V Figure 15). Thereafter, the cells were incubated for 24h before proceeding to total RNA isolation.
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Figure 15: Keratinocytes treated or not with plasma for 6 and 24h.
4.2. Isolation total RNA (Pure Link RNA Mini Kit Life Technologies)
The Pure Link RNA Mini Kit provides a rapid method for isolation high-quality total RNA from cells and other sources. The purified total RNA is suitable for sequencing experiments.
Samples are lysed and homogenized in the presence of guanidinium isothiocynate, a chaotropic salt capable of protecting RNA from endogenous RNases. After homogenization, ethanol is added to the sample. The sample is then processed through a spin cartridge containing a clear silica-based membrane to which the RNA binds. Any impurities are effectively removed by subsequent washing. The purified total RNA is then eluted in RNAse-free water (or Tris Buffer, pH7, 5).
STOP Samples can be stored at -20°C
Quality control by Agarose RNA-Gel electrophoresis: The quality control of the isolated RNA was done by visualizing 28S and 18S rRNA in an agarose gel. Running RNA in an agarose gel is quite different than running a DNA gel. RNA has the tendency to form secondary and tertiary structures that can impede its separation. Therefore, the agarose gel has to be prepared under denaturing condition by using a formaldehyde and MOPS (3-morpholinopropanesulfonic acid) buffer system.
Quantity control: The quantity of isolated RNA was determined by spectrophotometry UV absorbance by 260 nm. The quality of the isolated RNA is determined by the ratio OD260/280. A good quality RNA has a ratio of 1, 8 to 2, 0.
22
5. Preparation of the library
(Ion AmpliSeqRNA Library Kit, Life Technologies)5.1. Workflow
Figure 16: Workflow of the template preparation (Step 1)
5.2. Required materials and equipment
Ion Ampliseq RNA Library Kit
Ion Ampliseq RNA Custom Panels
Ion Xpress Barcode Adapters Kit
GeneAmp PCR System 9700 (Life Technologies)
Qubit 2.0 Fluorometer (Life Technologies)
Qubit dsDNA HS Assay Kit (Life Technologies)
Qubit Assay Tubes (Life Technologies)
PCR-tubes (0,2 ml)
Plate magnet
Nuclease-free Water
Ethanol (100 ml)
Pipettors 2-1000 µl
Low-retention pipette tips
Aim: Preparation of cDNA-library (targeted sequences) with barcode adapters
23 Table 1: List of required materials and equipment
Description Cap color Quantity Storage
Ion Ampliseq RNA RT Module
5x VILO RT Reaction Mix Red 32µl -20°C
10x SuperScript III Enzyme Mix Red 16µl -20°C
Ion Ampliseq RNA Dynabeads Cleanup Module
Binding Solution Concentrate Clear 2 ml RT
Wash Solution Concentrate Clear 2 ml RT
Dynabeads Magnetic Beads Red 200 µl 4°C (do not
freeze)
Nuclease-free Water Clear 1,75 ml 4°C
Processing Plate and Lid 1 RT
Ion Ampliseq Library Kit 2.0
5x Ion AmpliSeq HiFi Master Mix Red 32 µl -20°C
FuPa Reagent Brown 16 µl -20°C
Switch Solution Yellow 32 µl -20°C
DNA Ligase Blue 16 µl -20°C
Ion AmpliSeq Adapters Green 16 µl -20°C
Platinum PCR Supermix HiFi Black 400 µl -20°C
Library Amplification Primer Mix White 16 µl -20°C
Low TE Clear 1 ml RT
Ion AmpliSeq RNA Custom Panels
100 primer pairs per pool Clear 5x -20°C
Ion Xpress Barcode Adapters
Ion Xpress P1 Adapter Violet 320 µl -20°C
Ion Xpress Barcode <X> White 20 µl -20°C
5.3. Procedure
5.3.1. Reverse Transcription PCR
1. Combine the following components in a PCR-tube on ice. For multiple reactions, prepare a master mix without RNA.
Component Volume per rxn
5X VILO RT Reaction Mix 2 µl 10X Superscript III Enzyme Mix 1 µl Total RNA, 10ng <6 µl Nuclease-free Water to 10 µl
2. Gently mix the tube contents, and centrifuge the tube briefly to collect the contents at the bottom.
3. Load the tube in the thermal cycler, and run the following program to synthetize cDNA.
Temperature Time
42°C 30 min
85°C 5 min
4°C ∞
24 5.3.2. Amplify targets:
1. Combine the following components on ice, in a PCR tube:
Component Volume
5X Ion AmpliSeq HiFi Master Mix 4 µl
5X Ion RNA Panel 4 µl
Nuclease-free Water to 10 µl 2. Pipette the mixture up and down 5 times.
3. Add the entire contents of the reverse transcription reaction (now the total volume is 20 µl) and gently mix again.
4. Seal the tubes, load the samples in the thermal cycler and run the following program to amplify target cDNA region:
Stage Step Temperature Time
Hold Activate the enzyme 99°C 2 min
20 cycles Denature 99°C 15 sec
Anneal and extend 60°C 4 min
Hold - 10°C ∞
STOP Samples can be stored at 10°C overnight or at -20°C for longer periods
5.3.3. Partially digest primer sequences
1. Add 2 µl of FuPa Reagent to each amplified sample (Now the total volume is 22µl) 2. Pipette the mixture up and down 5 times.
3. Seal the tubes, load the samples in the thermal cycler and run the following program to partially digest the primer sequences:
Temperature Time
50°C 10 min
55°C 10 min
60°C 20 min
10°C Hold (for up to 1 hour)
5.3.4. Ligate adaptors to the amplicons and purify
1. For each barcode X chosen, prepare a mix of Ion P1 Adapter and Ion Xpress Barcode X.
Diluted barcode adapter mix
Component Volume
Ion P1 Adapter (violet cap) 2 µl Ion Ampliseq Barcode X (white cap) 2 µl
Nuclease-free water 4 µl
Store diluted adapters at -20°C.
2. Resuspend the switch solution by pipetting up and down.
25 3. Add following components to the digested sample (22µl):
Component Volume
Digested sample 22 µl
Switch Solution 4 µl
Diluted barcode adapter mix 2 µl
Total volume 28 µl
4. Add 2µl DNA Ligase (Total volume 30 µl) 5. Pipet up and down 5 times
6. Seal the tubes, load the samples in the thermal cycler and run the following program:
Temperature Time
22°C 30 min
72°C 10 min
10°C Hold
STOP Samples may be stored at -20°C 5.3.5. Purify the unamplified library:
- Complete the wash solution.
- Warm the binding solution concentrate at RT or 37°C, evtl. shake the solution - Incubate the Dynabeads Magnetic beads at RT for 10 min. Vortex before pipetting.
- Pre-heat the elution solution, Nuclease-free water or Low-TE at 37°C for 5 min.
First-round purification:
1. Vortex Dynabeads Magnetic beads, add 5 µl of beads in a new PCR-tube.
2. Add 72 µl of Binding Solution Concentrate, pipette up and down 10 times 3. Add 30 µl Nuclease-free water to your 30 µl unamplified library, mix gently.
4. Add the 60 µl unamplified library to the beads and binding Solution mixture, mix up and down 5 times.
5. Pre-wet a pipette tip by pipetting 32µl of 100% ethanol up and down 3 times.
6. without changing tips, add 32µl of 100% ethanol, mix up and down 10 times.
7. Incubate the sample for 5 min at RT.
8. Place the tube on the magnetic stand for 2 min until the solution is clear.
9. Carefully remove the supernatant and transfer to a fresh well.
Keep the supernatant! It contains your library!!
Second-round purification:
1. Vortex the Dynabeads Magnetic beads, add 5 µl of beads to the supernatant (about 162 µl) 2. Add 25 µl of Binding Solution Concentrate, mix up and down 5 times.
3. Pre-wet a pipette tip by pipetting 32µl of 100% ethanol up and down 3 times.
4. without changing tips, add 32µl of 100% ethanol, mix up and down 10 times.
5. Incubate the sample for 5 min at RT.
6. Place the tube on the magnetic stand for 2 min until the solution is clear.
7. Keeping the plate on the magnetic stand, carefully remove and discard the supernatant Keep the Beads! The library is bound to the beads!!
26
1. While the plate is on the magnetic stand, add 150 µl of completed Wash solution, incubate for 30 s.
2. Keeping the plate on the magnetic stand, carefully aspirate and discard the supernatant.
3. Air dries the beads at RT for 3 min.
4. Remove the plate from the magnetic plate, add 10 µl of pre-heated (37°C) Nuclease-free Water, pipet up and down 10 times.
5. Incubate 1 min.
6. Place the tube on the magnetic plate for 1 min.
7. Carefully remove the supernatant, which contains your library, transfer to a fresh tube, without disturbing the beads.
Keep the supernatant! It contains your library!!
5.3.5. Amplify the library and purify Amplify library
1. On ice, combine in a 0.2ml PCR tube:
Component Cap color Volume per rxn
Platinum PCR SuperMix HiFi black 50 µl
Library Amplification Primer Mix white 2 µl
Purified, unamplified library 10 µl
2. Seal the tubes; load the sampled in the thermal cyclers with following program:
Stage Step Temperature Time
Hold Activate the enzyme 98°C 2 min
5 cycles Denature 98°C 15 sec
Anneal and extend 60°C 1 min
Hold - 10°C ∞
STOP The amplified library may be stored at -20°C
Purify the amplified library
1. Vortex the Dynabeads magnetic beads thoroughly, and add 10 µl of the Dynabeads Magnetic Beads to a new tube
2. Add 72 µl of Binding Solution Concentrate, pipet up and down 10 times
3. Add 62 µl of your amplified library to the beads and Binding Solution Mixture, mix by pipetting up and down 5 times (Total volume 144 µl)
4.Pre-wet a pipette tip by pipetting 45 µl pf 100% ethanol up and down 3 times
5. Without changing tips add 45 µl of 100% ethanol and mix by pipetting up and down 10 times 6. Incubate the samples for 5 min at RT
7. Place the plate on the magnetic stand for 1-2 min until the solution is clear, to capture the beads.
8. Keeping the plate on the magnetic stand, carefully remove and discard the supernatant IMPORTANT! Keep the beads! The library is bound to the beads.
Wash the beads and elute the library
1. While the plate is on the magnetic stand, add 150 µl of the complete Wash Solution, and incubate the samples for 30 s.
27
2. Keeping the plate on the magnetic stand, carefully aspirate and discard the supernatant without disturbing the beads.
3. Air dries the beads at RT for 3min
4. Remove the plate from the magnetic stand, add 15µl of pre-heated (37°C) Low TE, and pipet up and down 10 times
5. Incubate 1 min at RT
6. Place the tube on the magnetic stand for 1min.
7. Keeping the plate on the magnetic stand, carefully remove the supernatant, containing the purified library, and transfer to a fresh tube, without disturbing the beads.
IMPORTANT! Keep the supernatant! It contains the desired library.
5.3.6. Quantify the library and dilute for the template preparation (Qubit dsDNA HS Assay Kit) 1. Make a 1:200 working dilution of Qubit dsDNA HS reagent using the Qubit dsDNA HS Buffer.
2. Prepare 1:10 dilution of the amplified library in Nuclease-free Water, and add 10 µl of the diluted library to 190 µl diluted dye reagent.
3. Prepare each Qubit standards:
4. Measure each Qubit Standard on the Qubit 2.0 Fluorometer 5. Calculate the concentration of the undiluted library.
6. Calculate the library dilution that gives a concentration of 15 ng/ml in a total volume of ca. 30 µl.
7. Dilute an aliquot of each library with low TE to a concentration of 15 ng/ml.
STOP Undiluted library may be stored at -20°C (>48h), otherwise 4°C. Store diluted libraries at 4°C (max.
48h).
5.3.5. Combine barcoded libraries from different samples
Combine equal volumes of barcodes libraries (for ex. untreated and plasma treated). They will be loaded on a single Ion Chip later on. The concentration of the library pool is 15 ng/ml.
28
6. Preparation of the sequencing template
(Ion PGM Template OT2 200 Kit)6.1. Workflow
Figure 17: Workflow of the template preparation before sequencing
6.2. Required materials and equipment
diluted library
Ion OneTouch System
PCR Thermo-Cycler
1,5ml Low-Bind Reactions tube
Microcentrifuge
Pipettors 2-1000 µl
Low-retention pipette tips
Vortexer
15ml Falcon tubes
50ml Falcon tubes
Heat block 75°C
Magnet
0,2ml PCR-tubes
1M NaOH
Aim: Preparation and enrichment of Ion Sphere Particles linked to amplicon.
29
Table 2: List of required materials and equipment
Kits Cap color Quantity Storage
Ion PGM Template OT2 Reactions 200 Kit Ion PGM One Touch Plus Reaction
Filter Assembly
10 RT
Ion PGM Template OT2 Supplies 200 Kit
Ion OneTouch Reagent tubes 2 RT
Ion OneTouch Recovery Tubes 20 RT
Ion OneTouch Recovery Routers 10 RT
Ion OneTouch Sipper Tubes 2 RT
Ion OneTouch2Amplification Plates 10 RT
Ion OneTouch ES Supplies 1 RT
Ion OneTouch 2 Cleaning Adapters 10 RT
Ion PGM Template OT2 Reagents 200 Kit Ion PGM Template OT2 200 Reagent
Mix
violet 5x, je 1000 µl -20°C, 4°C after thawing!
Ion PGM Template OT2 200 Enzyme Mix
Brown 500 µl -20°C
Ion PGM Template OT2 200 Ion Sphere Particles
Black 1000 µl -20°C
Ion PGM Template OT2 Solutions 200 Kit Ion PGM Template OT2 200 PCR
Reagent B
Blue 2x, je 1500µl RT
Ion OneTouch Oil 450 ml RT
Ion OneTouch Reaction Oil 27 ml RT
Nuclease-free Water 15 ml RT
Ion PGM OT2 Recovery Solution 350 ml RT
Ion OneTouch Wash Solution 20 ml RT
MyOne Beads Wash Solution Green 2x, je 1400 µl RT
Neutralization Rot 100 µl RT
Tween Solution 6 ml RT
Ion PGM Enrichment Beads Blue 150 µl 4°C
30
Figure 18: The IonTouch2 machine (from Life Technologies application note).
6.3. Procedure
6.3.1. Ion OneTouch™ 2 Instrument Set up the Ion OneTouch™ 2 Instrument
1. Install the Ion OneTouch™ Recovery Tubes and Ion OneTouch™ Recovery Router 2. Install the 2 Recovery Tubes and the Recovery Router, and then close the centrifuge lid.
Install the Ion OneTouch™ 2 Amplification Plate
1. If there is a used cleaning adapter on the instrument, remove and appropriately discard it. Open the heat block.
2. Hold the disposable injector in one hand and the Amplification Plate in the other hand.
3. Insert the Amplification Plate into the heat block, then close the heat block and thread and install the disposable tubing.
Install the disposable injector
1. Insert the disposable injector until the injector stops at the router base. Release the disposable injector.
2. Confirm automatic placement of the disposable injector by pressing then releasing the spring- loaded top of the Injector Hub.
3. Note: You should hear a click and the Injector Hub should return to the up position.
Install the reagents
1. If you are using a new kit, appropriately discard of the Recovery Solution and used Reagent Tubes and contents.
2. Use fresh gloves to install new sipper tubes.
3. Install Ion OneTouch™ Oil on the left front port: invert the bottle 3 times, fill the Reagent Tube half- full, and install the tube.
31
4. Install Recovery Solution on the right front port: invert the bottle 3 times, fill the Reagent Tube quarter-full, and install the tube.
Empty the waste container and the oil waste tray
1. Empty the waste container and reattach the tubing to the container.
2. Slowly pull out, but do not remove, the oil waste tray. If there is no oil, reinsert the tray. Dispose of any excessive oil.
Prepare the amplification solution (in a pre-PCR dedicated room)
1. In general, Ion Ampliseq libraries at 100pM (or 15 ng/ml) yield sufficient monoclonal Ion Sphere Particles (ISPs). Dilute your library: to 21 µl Nuclease Free water, add 4 µl library (Total volume 25 µl). Vortex 5 seconds, centrifuge 2 seconds, place on ice.
2. Prepare the Reagent Mix, PCR Reagent B, Enzyme Mix, and library.
3. In a 1.5-mL Eppendorf LoBind® Tube, add the following components in order and pipet up and down after adding each component:
Order Reagent Cap color Volume
1 Nuclease-Free Water - 25 µl
2 Ion PGM Template OT2 200 Reagent Mix
Violet 500 µl RT, vortex 30s then centrif.
(store later at 4°C) 3 Ion PGM Template OT2 200 PCR
Reagent B
Blue 300 µl RT, Vortex 1 min, and then centrif.
4 Ion PGM Template OT2 200 Enzyme Mix
Brown 50 µl Centrifuge 2s, place on ice 5 Freshly prepared, diluted library
(not stock library)
- 25 µl On ice
Total volume 900 µl
4. Vortex 5 seconds, centrifuge 2 seconds.
5. Prepare the Ion PGM™ Template OT2 200 Ion Sphere™ Particles (ISPs): Vortex the Ion PGM™
Template OT2 200 Ion Sphere™ Particles at maximum speed for 1 minute to resuspend the particles.
6. Add 100µl Ion PGM™ Template OT2 200 Ion Sphere™ Particles to the amplification solution, vortex for 5 seconds.
7. Place a Plus Reaction Filter Assembly into a tube rack so that the 3 ports of the filter face up.
8. Vortex the amplification solution at maximum speed for a full 5 seconds and centrifuge the solution for 2 seconds.
4. Slowly pipet the entire 1000 μl of solution through the sample port.
5. Slowly pipet 1000 μl of Ion OneTouch™ Reaction Oil into the sample port; change tip, then slowly add another 500 μl of oil.
6. Invert Reaction Filter Assembly: Rotate the assembly to your right until the reaction tube is inverted and the 3 ports face down.
7. Firmly insert the 3 ports into the three holes on the top of the Ion OneTouch™ 2 Instrument.
Run the Ion OneTouch 2 Instrument
1. Ensure that the centrifuge lid of the Ion OneTouch™ 2 Instrument is closed. Touch Run.
2. Touch the drop-down menu, and then select PGM: Ion PGM™ Template OT2 200 Kit. Touch Next.
3. Choose Assisted or Expert. Complete the listed tasks, and then touch Next.
32
Recover the Ion PGM™ Template OT2 200 Ion Sphere™ Particles
1. At the end of the run, follow prompts to centrifuge the sample if more than 15 minutes have elapsed since the end of the run.
2. After the centrifuge stops, touch Open Lid, wait until the lid opens, then remove and discard the Recovery Router.
3. Carefully remove both Recovery Tubes, and put the tubes in a rack.
4. Remove and discard all but 50 μl of the Recovery Solution from each Recovery Tube without disturbing the pellet.
5. With a new tip, resuspend the ISPs in the remaining Recovery Solution by pipetting the pellet up and down.
Quality control using Qubit 2.0 Fluorometer
Assess the quality of a 2 µL aliquot of the unenriched, template-positive ISPs using a Qubit® 2.0 Fluorometer. See Ion PGM Template OT2 Kit User Guide Page 68
To store the ISPs: combine suspensions, add 1 mL of Ion OneTouch Wash Solution, and store at 4°C.
6.3.2. Enrich template-positive OT2 200 Ion Sphere™ Particles with Ion OneTouch™ ES
Prepare reagents
Prepare fresh Melt-Off Solution (125 mM NaOH, 0, 1% Tween 20) by combining the following reagents in this order:
Order Component Volume
1 Tween Solution 280 µl
2 1 M NaOH 40 µl
Total 320 µl
Wash and resuspend Dynabeads® MyOne™ Streptavidin C1 Beads
1. Vortex the tube containing Dynabeads® MyOne™ Streptavidin C1 Beads for 30 seconds, then centrifuge the tube for 2 seconds.
2. Open the tube, and then use a new tip to pipet up and down the dark pellet of beads until the pellet disperses.
3. Transfer 13 μl of the beads to a new 1.5-mL Eppendorf LoBind® Tube.
4. Place the tube on a magnet for 2 minutes, then carefully remove and discard the supernatant without disturbing the pellet.
5. Add 130 μl of MyOne™ Beads Wash Solution to the beads.
6. Remove the tube from the magnet, vortex the tube for 30 seconds, and centrifuge the tube for 2 seconds.
Fill the 8-well strip
1. Obtain an 8-well strip from the Ion OneTouch™ ES Supplies Kit. Ensure that the square-shaped tab of an 8-well strip is on the left.
2. Obtain suspensions of template-positive Ion Sphere Particles (ISPs), prepared as described in the Ion PGM™ Checklist — Ion OneTouch™ 2 Template Preparation (MAN0009128).
33
3. If ISPs were stored at 2–8°C, centrifuge (15,500 × g for 2.5 minutes), remove all but 100 μl, and resuspend (pipet up and down).
4. Transfer the ISP suspensions from both tubes (1 tube if stored) into Well 1 of the 8-well strip (total of 100 μl in the well).
5. Fill the remaining wells as follows, then immediately proceed to step 6:
Well number Reagent to dispense in well
Well 1 (next to the square tab) Entire template-positive ISP sample (100 µl)
Well 2 130 µl of Dynabeads MyOneTM Streptavidin C1
Beads resuspended in MyOneTM Beads Wash Solution
Well 3
300 µl of Ion OneTouchTM Wash Solution (W) Well 4
Well 5
Well 6 Empty
Well 7 300 µl of freshly-prepared Melt-Off solution
Well 8 Empty
Confirm that the square-shaped tab is on the left, and then insert the filled 8-well strip pushed to the far right end of the tray slot.
Prepare the Ion OneTouch™ ES
1. Place a new tip in the Tip Loader, and press the Tip Arm firmly down onto the tip to load the tip.
2. Return the arm with the installed tip to its cradle.
3. Add 10 μl of Neutralization Solution to a new 0.2-mL PCR tube.
4. Insert the opened 0.2-mL PCR tube with the Neutralization Solution into the hole in the base of the Tip Loader.
Perform the run
1. Confirm that the new tip and 8-well strip have been correctly loaded.
2. Pipet the contents of Well 2 up and down to resuspend the beads before starting the run. Do not introduce bubbles into the solution.
3. If necessary, turn ON the Ion OneTouch™ ES. Press Start/Stop to begin the run.
4. Immediately after the run, close and remove the PCR tube containing the enriched ISPs. Press Start/Stop to reset the instrument.
5. Ensure the 0.2-mL PCR tube contains >200 µL and mix the contents of the tube by gently inverting the tube 5 times.
6. Remove and discard the used tip by twisting the tip counterclockwise and pulling it downward.
7. Remove and discard the used 8-well strip.
8. Proceed to sequencing using the Ion PGM™ Sequencing 200 Kit v2 (Cat. no. 4482006) or store the material.
Store the material at 4°C for up to 3 days.
34
7. Sequencing
(Ion PGM Sequencing 200 Kit v2, Life Technologies) Aim: Massive parallel sequencing by synthesis of all amplicons7.1. Workflow
Figure 19: Workflow of the sequencing process
7.2. Required materials and equipment
Ion314TM Chip Kit v2 (Life Technologies)
Ion Personal Genome Machine (PGM) System (Life Technologies)
Torrent Server (Life Technologies)
Nitrogen Gas canister
18mOhm water
Ion Chip Minifuge (Life Technologies)
Microcentrifuge (16.000g)
0,22µm Filter
Pipette 2-1000 µl
Low-retention filtered pipette tips
PCR-tubes, flat cap, 0,2ml
Vortexer
Thermal cycler
Cylinder (1L or 2L)
1L bottle
15ml tubes
NaOH (10M)
1,5ml Eppendorf tubes
1M HCl solution
Table 3: List of required materials and equipment
Kits Color Quantity Volume Storage
Ion PGM Sequencing Supplies 200 v2 Wash Bottle Sipper Tubes Gray
8 tubes for a 250ml bottle 4 tubes for a 2L bottle
- RT
Reagent Bottle Sipper Tubes Blue 16 tubes - RT
Reagent Bottles w/ labels (50 mL) - 25 bottles - RT
35
Wash 1 Bottle w/ label (250 mL) Green 1 bottle - RT
Wash 2 Bottle w/ label (2 L) Green 1 bottle - RT
Wash 3 Bottle w/ label (250 mL) Green 1 bottle - RT
Ion PGM Sequencing Reagents 200 v2
Ion PGM Sequencing 200 v2 dGTP Black 1 100 µl -20°C
Ion PGM Sequencing 200 v2 dCTP Blue 1 100 µl -20°C
Ion PGM Sequencing 200 v2 dATP Green 1 100 µl -20°C
Ion PGM Sequencing 200 v2 dTTP Red 1 100 µl -20°C
Ion PGM Sequencing 200 v2 Polymerase Yellow 1 36 µl -20°C
Sequencing Primer White 1 144 µl -20°C
Control Ion Sphere Particles Clear 1 60 µl -20°C
Ion PGM Sequencing Solutions 200 v2 Ion PGM Sequencing 200 v2 W2
Solution Black 4 bottles 126,25 ml
each
4°C Protect from
light!
Ion PGM Cleaning Tablet - 4 - 4°C
Annealing Buffer - 1 bottle 4°C
Ion PGM Sequencing 200 v2 1x W3
Solution - 2 bottles 4°C
Figure 20: The PGM sequencing machine.
7.3. Procedure
7.3.1. Initialize the Ion PGM™ System (about 1 hour) Before initialization
1. Thaw dNTP stock solutions on ice.
2. Check the nitrogen gas tank; change if the pressure drops below 500 psi.
Prepare the Wash 2 Bottle
1. Rinse the Wash 2 Bottle three times with 200 mL of 18 MΩ water.
2. Fill the bottle with 18 MΩ water to the appropriate mold line.
3. Add the entire bottle of Ion PGM™ Sequencing 200 v2 W2 Solution to the Wash 2 Bottle.
36
4. Add 70 μl of freshly prepared 100 mM NaOH solution to the Wash 2 Bottle.
5. Cap the bottle and invert five times to mix, and immediately proceed through the rest of the initialization procedure.
Prepare the Wash 1 and Wash 3 Bottles
1. Rinse the Wash 1 and Wash 3 Bottles three times with 50 mL of 18 MΩ water.
2. Add 350 μl of freshly prepared 100 mM NaOH to the Wash 1 Bottle and cap the bottle.
3. Add Ion PGM™ Sequencing 200 v2 1X W3 Solution to the 50-mL line marked on the Wash 3 Bottle and cap the bottle.
Begin the initialization
1. Confirm that the chip used for cleaning is still in place on the instrument.
2. Press Initialize on the Ion PGM™ Sequencer touchscreen.
3. Scan the barcode on the W2 Solution bottle or select Ion PGM™ Sequencing 200 Kit v2 on the touchscreen.
4. Press Next and confirm that sipper tubes and collection trays are in place. Press Next to verify the gas pressure.
5. If the gas pressure is sufficient, press Next. If the pressure is low, press Yes to re-verify.
6. Wearing clean gloves, insert a new, long sipper tube into the cap in the W2 position.
7. Immediately attach the prepared Wash 2 Bottle and tighten the cap. Press Next.
8. Change gloves. Install new, short sipper tubes in the caps in W1 and W3 positions.
9. Immediately attach the prepared Wash 1 and 3 Bottles and tighten the caps. Press Next to begin initialization.
Prepare the 50-mL Reagent Bottles with dNTP solutions
1. Vortex each thawed dNTP stock solution and centrifuge to collect the contents.
2. Label four new Reagent Bottles as dGTP, dCTP, dATP, and dTTP.
3. Transfer 20 μl of each dNTP into its respective Reagent Bottle.
4. Attach the sipper tubes and Reagent Bottles
5. After the wash solutions have initialized, remove the used sipper tubes and collection trays from the dNTP ports.
6. Using new gloves, attach a new short sipper tube to each dNTP port.
7. Attach each Reagent Bottle to the correct dNTP port and tighten until snug.
8. Follow the touchscreen prompts to complete initialization.
9. Proceed to the appropriate sequencing protocol for your chip type.
7.3.2. Sequencing
-Before starting, thaw Sequencing Primer on ice.
-Add Control Ion Sphere Particles (IPSs) to the enriched, template-positive ISPs.
1. Ensure that enriched, template-positive ISPs (prepared using the Ion PGM™ Template OT2 200 Kit) are in a 0.2-mL non-polystyrene PCR tube.
2. Vortex Control ISPs and centrifuge for 2 seconds.
3. Add 5 μl of Control ISPs to the appropriate volume of enriched, template-positive ISPs for your chip type: Here Ion314TMChipv2:
Half the volume of prepared ISPs (transfer and store half of the volume)
37 Anneal the Sequencing Primer
1. Mix the tube containing ISPs and controls by thoroughly pipetting up and down. Centrifuge for 2 minutes at 15,500 × g.
2. Carefully remove the supernatant, leaving 3 µl volumes in the tube.
3. Add Sequencing Primer and confirm the total volume (adjust the volume with Annealing Buffer, if necessary):
Volume Ion314TMChip v2
Sequencing Primer 3 µl
Total Volume 6 µl
4. Pipet the sample up and down thoroughly to disrupt the pellet.
5. Program a thermal cycler (95ºC for 2 minutes, then 37ºC for 2 minutes), insert tube in cycler, and run the program.
1. Remove a new chip from its package and label it. Press Run on the Ion PGM™ Sequencer touchscreen.
2. When prompted, ground yourself. Replace the old chip in the chip clamp with the new chip. (Do not wear gloves.)
3. When prompted, scan the new chip package barcode or press Change to manually enter barcode.
Press Chip Check.
Volume Ion 314TM Chip v2
Ion PGM Sequencing 200 v2
Polymerase 1 µl
Total Volume 7 µl
2. Pipet the ISPs up and down, and incubate at room temperature for 5 minutes.
Load the chip
1. Tilt the new chip 45 degrees. Insert pipette tip into the loading port; remove and discard as much liquid as possible.
2. Place the chip upside-down in the Ion Minifuge bucket; transfer the bucket to the Minifuge (chip tab pointing in).
3. Centrifuge for 5 seconds. Remove the chip from the bucket and wipe off any liquid on the bucket.
4. Put the chip back in the bucket and place the bucket on a flat, stable surface.
5. Following polymerase incubation, collect the entire sample into the pipette tip and insert the tip into chip loading port.
6. Dial down the pipette to gently and slowly deposit the ISPs, leaving ~0.5 μl of sample in the pipette tip.
7. Remove and discard any displaced liquid from the other port of the chip.
8. Transfer the chip in the bucket to the Minifuge (chip tab in). Centrifuge for 30 seconds.
9. Set the pipette volume according to the following table:
Volume Ion314TMChip v2
Pipette setting 5 µl
38
10. Tilt the chip 45 degrees, and slowly pipet the sample in and out of the chip three times.
11. Centrifuge the chip in the Minifuge (with the chip tab out) for 30 seconds.
12. Repeat step 10 one more time, then centrifuge for 30 seconds (chip tab in).
13. Tilt the chip and slowly remove liquid by dialing the pipette.
14. Discard the liquid. If some liquid remains in the chip, perform a 5-second quick spin (chip tab out).
15. If necessary, lightly tap the point of the chip tab against the benchtop, and remove and discard any collected liquid.
Select the Planned Run and perform the run
1. Press Browse on the sequencer touchscreen and select the name of the plan you created. Press Next.
2. Confirm that the run settings are correct or make any necessary changes. Press Next.
3. Load and clamp the chip, then press Next.
4. Visually inspect the chip in the clamp for leaks before closing the cover. If the chip passes, press Next.
5. If it fails, press Abort, re-seat chip, and press Calibrate.
6. If the chip continues to fail calibration, contact Technical Support.
8. Check Run Report and insight into Data Analysis
8.1. Check Run Report
The information about bases and reads are shown in the Run Report. Before initiating the process of sequence mapping, we will analyze the run report.
The sequencing run should be evaluated according to the total number of base sequenced. This depends on the loading density, the total number of reads (usable sequence) and the read length.
Figure 21: Run report: summary of unaligned reads. The ISP density image shows percent loading across the surface of the ion Chip.
