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INOM

EXAMENSARBETE BIOTEKNIK, AVANCERAD NIVÅ, 30 HP

STOCKHOLM SVERIGE 2016,

Development of a massive parallel sequencing method for population genetics, for the sequencing of 1,000 dog mitochondrial genomes per Miseq run, based on nested and multiplexed PCR amplification and PCR-incorporated dual-index identification barcodes

LINNEA GULDBRAND

KTH

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Development of a massive parallel sequencing method for population genetics, for the sequencing of 1,000 dog mitochondrial genomes per Miseq run, based on nested and multiplexed PCR amplification and PCR-incorporated

dual-index identification barcodes

Linnea Guldbrand

Master Thesis at the School of Biotechnology, KTH Royal Institute of Technology,

Department of Gene Technology, SciLifeLabs

Supervisor and Examiner: Peter Savolainen

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Abstract

The geographical origin of the domestic dog has not yet been conclusively established. The mitochondrion, being matrilineally inherited and prone to a greater rate of mutation compared to nuclear DNA, is of great significance in genetic evolutionary studies and as such, complete sequencing of the mitochondrial genome of a great number of individuals would provide important data for the furthering of such studies.

This project aims to design a method of sequencing the entire mitochondrial genome of the domestic dog for a large number of individuals in parallel on the Illumina MiSeq sequencing platform, using several sets of PCR primers to generate barcoded and sequencing-ready libraries of predetermined fragments for each individual.

PCR primers were constructed both for initial long-range products and for shorter fragments, suitable for sequencing and containing partial sequencing adaptors, covering the entire mitochondrial chromosome. Additionally, primers containing barcode indices and the final required sequencing constructs were designed. The viability of the primers and of different PCR parameters were investigated, verified on agarose gels and Bioanalyzer, and a set of samples were taken through cleaning, barcoding, and sequencing.

Results indicate a promising method, where all primers successfully generate product, and both cleaning and sequencing appears in essence successful, but the relative amounts of product obtained from each primer, and subsequently the amount of reads obtained in sequencing, varies significantly with the initial set up. Subsequent experiments, performed after the closing of the practical part of the project, have shown that compensating for this uneven amplification by using significantly unequal primer concentrations greatly serves to alleviate these issues.

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Abstract ... 2

Introduction ... 4

Previous Findings on the Geographical Origins of the Domestic Dog ... 4

The Mitochondrion, in Biology and in Forensics ... 5

The Illumina, Dual Indexed, Paired End Sequencing Method ... 6

Existing Methods for Whole mtDNA Sequencing ... 9

Aim of Project ... 10

Materials and Methods ... 11

Primer Layout and Design ... 11

PCR Reactions ... 11

Results ... 13

Primer Layout and Design ... 13

PCR Reactions, Viability of Primers and Multiplex Set Ups ... 20

Barcoding PCR ... 34

Concentration Measurements and Cleaning ... 35

Initial Sequencing ... 37

Results Obtained Post-Project ... 40

Discussion ... 41

Primer Layout and Design ... 41

PCR Procedures ... 43

Indexing, Cleaning, and Sequencing ... 44

Conclusions ... 44

References ... 46

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Introduction

Previous Findings on the Geographical Origins of the Domestic Dog

That the domestic dog has its evolutionary origin in the wolf has long been known and

accepted as fact, based on both genetic evidence and on archaeological findings, as well as on behavioural and physical traits [1]. However, the precise circumstances, the historical time point, and the geographical location of the original domestication event, or events, are less clear. Several evolutionary genetic studies have been performed, using various methods and sample materials, with varying results. Simply put, these studies attempt to identify the most likely common ancestor of the domestic dog as the one whose genetic material can account for the diversity of all others, while also comparing them to wild wolf populations and estimating the timeframe for the domestication event via the rate at which mutations are believed to accumulate. Variously, such studies have indicated the geographical origin of the domestic dog in places as disparate as Europe, South East Asia, and the Middle East.

A 2002 study [1] of a stretch of 582 base pairs from the so-called control region of the mitochondrial DNA from 654 dogs, representing dog populations worldwide, indicated an East Asian origin for the domestic dog, based on a comparatively higher degree of

phylogenetic variation in dogs from this area. These findings were corroborated by the analysis of 14 437 base pairs from the Y chromosome (fragmented sequences from incomplete sequencing of a male dog DNA, assigned to the Y chromosome through

comparison to female dog DNA as well as the human Y chromosome sequences [21]) from 151 dogs worldwide [2] as well as by a further study of the complete mitochondrial DNA from 169 individuals, combined with the 582 control region base pairs from 1576 individuals, both placing the geographical origin of the domestic dog in South Eastern Asia, south of the Yangtze River, China, less than 16 300 years ago [3]. Both studies also show that this region of South East China, south of the Yangtze River, is the region in which the genetic diversity is the greatest, and is the only region where almost all haplotypes of both the mtDNA and the Y chromosome can be found simultaneously. Additionally, analysis of the mtDNA of Native American dog breeds, when compared to East Asian and European dogs, as well as Pre- Columbian samples, show low levels of European mtDNA [4], indicating a more ancient, Asian, origin of the Native American dog breeds. Similarly, mtDNA analysis of the Australian Dingo and Polynesian domestic dogs indicate an origin in mainland South East Asia [5].

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On the other hand, a study of the mitochondrial DNA from 18 ancient canids [6] indicated a closer relationship with either ancient or modern European canids for all modern dogs the world over. The study did, admittedly, lack ancient canid samples from both the Middle East and China, two other major candidates for the geographical origin of the domestic dog.

Furthermore, genome-wide SNP (Single Nucleotide Polymorphism) analysis of over 48,000 SNPs in 912 dogs as well as 225 grey wolves has indicated a Middle Eastern origin for the domestic dog, based on the significantly larger genetic variation found in breeds from this region [7].

The Mitochondrion, in Biology and in Forensics

The mitochondrion is an organelle present in eukaryotic cells, whose role is to perform oxidative metabolism, providing energy for the cell. The origin of the mitochondrion is assumed to be the enveloping of a purple bacterium by an ancient eukaryotic ancestor, resulting in an endosymbiotic relationship whereby both the eukaryotic host cell and the bacterium symbiont benefits. Certain genetic material has since migrated from the original bacteria into the nucleic DNA of the host cell to the degree that the modern mitochondrion can no longer survive independently, but does retain certain vital genes in its own,

mitochondrial DNA, the mtDNA. They also still, like bacteria, reproduce through division, rather than through being disassembled and reassembled, as is the case with all other organelles, apart from the chloroplasts of plants, which have similar origins to the mitochondrion. [8]

Mitochondrial DNA is usually comprised of one essential double-stranded, circular chromosome, in multiple copies, but single-stranded, and linear chromosomes exist. The mitochondrial DNA encodes for components necessary for protein production and certain enzymes required for aerobic metabolism, but many components of the mitochondrion are encoded by nuclear DNA and are transported into the mitochondrion. Multiple copies of mitochondria are present in any given cell, and their genetic make up are not necessarily homogenous. [8]

Mitochondria are inherited maternally. In meiosis in females, mitochondria are evenly segregated between the two new cells and in the resulting embryo, the mitochondria present in the fertilised ovum will divide and produce all the mitochondria in the new organism. Due to this manner of inheritance, mitochondrial DNA can be used in forensics, to trace familial

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relations on the maternal side (e.g. mother and child, as well as siblings who share a mother, but not fatherhood), rule out suspects based on crime scene DNA, and on a larger time scale, trace the evolution of a species. Mitochondrial DNA is more suited to such analyses for two main reasons. Firstly, while each cell only contains one complete set up of nuclear DNA, mitochondrial DNA is present in multiple copies per cell, thereby making it significantly more abundant than nuclear DNA, somewhat circumventing the common issue of limited sample material. Secondly, specifically in mammals, mutations accumulate at a much higher rate in mitochondrial DNA than in nuclear DNA, on average 10-8 times per nucleotides and year, meaning that evolutionary differences can be visible on a comparative shorter timescale.

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The Illumina, Dual Indexed, Paired End Sequencing Method

The Illumina sequencing method is a Sequencing-By-Synthesis (SBS) sequencing method, known as Solexa, utilising fluorescently labelled nucleotides to track base incorporation. In its basic iteration, DNA is sheared into randomly sized fragments, to the ends of which a forward and a reverse adaptor sequence are ligated. These adaptor-ligated fragments are then, single- strandedly, randomly attached to the surface of the flow cell, where each individual fragment is amplified into clusters of multiple copies of the same fragment, using so-called bridge amplification. This means that the non-attached adaptor sequence of any given fragment anneals to its complementing adaptor on the surface of the flow cell, which then acts as a primer, allowing amplification into an arch-shaped double stranded structure where each of the two strands is in one end attached to the flow cell. A denaturation step separates the two strands of the ‘bridge’, and the process is repeated, until a sufficiently dense cluster of single stranded DNA fragments has been formed.

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Figure 1: Basic overview of Illumina sequencing, using random fragmentation and adaptor ligation [9]

After the clusters have been formed, the sequencing commences. Bases are determined through the use of fluorescently labelled nucleotides, each of the four bases fluorescing at a different wavelength. The labelled nucleotides are also reversible terminators, meaning that the fluorescent label blocks more than one nucleotide from being incorporated at a time, but that after the base has been determined, this label is enzymatically cleaved off, in preparation for the next cycle, allowing the next nucleotide to be incorporated. For the first cycle of the sequencing, all four fluorescently labelled nucleotides are added to the flow cell at once, together with primers specific to the adaptor sequences and DNA polymerase, and one nucleotide is incorporated in the first position of each strand, in each cluster. A laser is then used to excite the fluorescent label on the nucleotide, and its identity is recorded for each cluster. The label is then cleaved off, and all remaining reagents are washed away. For all subsequent cycles, all four labelled nucleotides and DNA polymerase is added, one base is incorporated in each strand in each cluster, laser excitation and image recording is performed, the label is cleaved off. Remaining reagents are then washed away in preparation for the next cycle. [9] [10]

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Figure 2: Sequencing-by-Synthesis using Illumina sequencing, by annealing one base at a time and detecting them by their fluorescent label [9]

This sequencing method provides sequencing data from all fragments applied to the flow cell, but has the downside of not being able to differentiate between the origins of the different fragments, as well as, depending on the size of the fragments, not being able to obtain full sequences, due to limitations imposed by the inherent read length of the sequencing method.

A way to enable the former is to employ the Illumina Single- or Dual-Indexed Sequencing method, both based on the Paired End Sequencing method. The second can be achieved by ensuring that all fragments used in the sequencing are below the maximum read length for the particular sequencing method.

The Dual-Indexed Paired End Sequencing method employs several modifications to the original adaptor construct and the sequencing procedure in order to distinguish between the origins of different sequenced fragments. Instead of a simple adaptor on each end of the fragment to be sequenced, the adaptor sequences are composed of several different

components each. These complex adaptors are shown in Figure 3, in a step-by-step depiction of the sequencing process. Upstream of the DNA insert to be sequenced, the components of the construct are the P5 adaptor, one of the slide-attaching sequences, which is also

complementary to the i5 Index Sequencing Primer, followed by the i5 Index, and the

sequence complementary to the Read 1 Primer, which initiates the sequencing from one end

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of the DNA Insert. Downstream, the DNA insert is followed by a stretch of bases that is complementary to both the i7 Index Sequencing Primer and to the Read 2 Primer, the i7 Index, and finally the P7 adaptor sequence that also attaches to the surface of the flow cell.

Each of the two index sequences is composed of 8 bases.

The sequencing includes four different primers, sequencing the DNA insert from both ends as well as the two indices. First, Read Primer 1 is aligned and the DNA insert is sequenced from the P5 end of the construct. The Read 1 product is then removed. Secondly, the i7 Index Sequencing Primer is used to sequence the i7 Index, after which the index product is

removed. Then, the P5 adaptor is annealed to its corresponding adaptor, grafted to the surface of the flow cell, which is used as the primer for the i5 Index. The i5 Index product is removed and the full complementary strand is generated and the original strand is removed. Lastly, Read Primer 2 is used to sequence the DNA insert form the P7 end. [11]

Figure 3: Schematic overview of the Dual-Indexed Paired End sequencing method, showing the order and the orientation of the primers involved [11]

Existing Methods for Whole mtDNA Sequencing

There are existing methods for sequencing the entire mitochondrial DNA of several individuals in parallel, using different set ups. One such is the PTS (Parallel Tagged Sequencing) method on the 454 sequencing platform [23, 24], using single-stranded, self- hybridising barcodes to tag samples prior to pooling and sequencing. Samples are barcoded separately and then pooled and prepared for sequencing. The barcodes are 6 bp long and

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allow for 72 samples to be sequenced in parallel. Another method is the PCR-product capture method [25], using fragments from a reference individual, fixed to beads, in order to retrieve and enrich mtDNA fragments from complex DNA mixtures. Long range PCR is used to produce two PCR fragments that cover the entire mtDNA, and these are then sonicated into 15-800 bp fragments, which are biotinylated and immobilized on streptavidin-coated beads.

The beads are then used to extract mtDNA fragments from sheared DNA mixtures, by

hybridisation, and the fragments can then be eluted, amplified, and sequenced, after separately barcoding each library and preparing them for sequencing.

Aim of Project

The aim of this project was to design and implement a method for the sequencing of the canine mitochondrial genome, for the purpose of producing data for phylogeographical analysis of the geographical origin of the domestic dog, for which large numbers of samples are necessary. The strategy employed was the introduction of barcodes during preparatory PCR in order to enable multiplexed sequencing, on the Illumina MiSeq, of 1152 individuals in parallel. Ultimately, the samples intended for use are saliva samples stored on FTA cards (Whatman).

In contrast to existing methods, the focus of this project lies on a high degree of

parallelisation, requiring steps taken to reduce workload and on streamlining the procedures, and on the specificity of the amplified fragments, in size and location, to guarantee the coverage of the entirety of the mtDNA, in fragments that can be fully sequenced by the Illumina MiSeq sequencing platform. The PTS method, being on the 454 sequencing platform and only providing a 72-plex, is therefore not suitable. Neither is the PCR-product capture method, both due to the fact that the intended sample material for the project is immobilised on FTA cards, and because it requires one library per individual to be prepared all the way to sequencing separately, which is both labour and cost intensive.

In order to enable these high degrees of parallelisation, it is important that the read numbers obtained from each fragment are as even as possible. This is to ensure that all fragments are sequenced with a sufficiently high redundancy to provide reliable output data.

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Materials and Methods

Primer Layout and Design

Primers were designed using the NCBI Primer BLAST tool [15], which can be used to generate primers according to a set of user specified parameters regarding, using the canine mtDNA reference genome [13] as the template.

The goal was to generate primers that would allow for the sequencing of the entire canine mitochondrial genome in fragments of a size that would be fully covered by the MiSeq sequencing platform. The highest number of base pairs the MiSeq can cover is 600 bp, which influences the number of primer pairs that are needed. These primers would, apart from the sequence-specific component, contain parts of the adaptor constructs necessary for MiSeq sequencing, to which barcoding primers, containing the rest of the necessary adaptors, can later be incorporated.

In addition to these primers, a set of primers, to be used for initial amplification of longer fragments, were desired. The purpose of these long-range primers are to limit the use of the original samples, to avoid depleting it, as well as to create a type of nested PCR [26] for the sequencing-specific fragments, reducing the likelihood of unspecific targets being generated by limiting the available unrelated template.

PCR Reactions

PCR reactions were carried out using either TagTaq, obtained from the Alba Nova University Center [22], or PlatinumTaq, produced by Invitrogen, both being polymerase enzymes for the purpose of the replication of DNA. The TagTaq was used for the inner primers, due to its availability and lower cost, as its lower processivity was deemed sufficient for the shorter inner primers, while PlatinumTaq was required to fully amplify the longer outer primers.

Originally, TagTaq was intended for both the inner and the outer primers, but after attempting to amplify the outer primers using the TagTaq, in multiple reaction set-ups, and failing to obtain product, possibly due to the outer fragments being too long for the TagTaq enzyme to successfully amplify, PlatinumTaq was employed instead.

The TagTaq-based reaction mixture consisted of 2.5 µl “P” (10x polymerase buffer, final concentrations 50 mM KCl, 2 mM MgCl2, 10 mM TrisHCl pH 8.5, 0.1% v/v Tween), 2.5 µl

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“C” (10x dNTP mix, containing 2 mM of each dNTP in water, final concentration 0.2mM), 1 µl Forward primer (0.2 µM final concentration), 1 µl Reverse primer (0.2 µM final

concentration), 1 µl template, and 17 µl nuclease-free H2O, to a final volume of 25 µl per reaction. Initially, 0.1 µl TagTaq was used per reaction, according to suggestions from the providers of the enzyme, but this was later increased to 0.2 µl per reaction due to the low yield.

For PlatinumTaq-based reaction mixtures, used for amplifying the outer fragments, volumes were adapted from the information sheet provide by Invitrogen [16] and consisted of 5 µl 10x PCR Buffer without MgCl2, 5 µl dNTP mixture (2 mM of each dNTP), 1.5 µl MgCl2 (50 mM), 2 µl Forward primer (0.2 µM final concentration), 2 µl Reverse primer (0.2 µM final concentration), 1 µl template, and 33.5 µl nuclease-free H2O, to a final volume of 50 µl per reaction. A volume of 0.2 µl PlatinumTaq, 5U/µl, per reaction was used throughout the experiments.

For both TagTaq-based and PlatinumTaq based reactions, in the case of multiplexing,

initially, equal amounts of each of the necessary forward and reverse primers were added, and the volume of H2O was lowered accordingly. In later experiments, in attempts to obtain comparable levels of each product in these multiplexes, the concentrations of the primers included in each multiplex were varied, increasing the concentration of those primers that failed to yield product in relation to those that did.

The PCR reactions were tried out with several different annealing temperatures, extension times, and numbers of cycles. The initial set up for the inner primers was 1.5 minutes of initial denaturation at 94°C, followed by 30 cycles of 30 seconds of annealing at 46°C and 2 minutes of extension at 72°C, a final extension for 10 minutes at 72°C and ending in a Hold at 4°C.

The number of cycles was later increased to 40, and both 49°C and 52°C as annealing temperatures were evaluated.

The PCR reaction parameters for the outer primers were initially the same as for the inner primers, with the exception of the annealing temperature being set to 50°C. This was later adjusted to evaluate both 5 and 10 minutes of extension time, as well as different numbers of cycles.

The annealing temperatures were chosen by manually calculating the optimal annealing temperature for each primer, using only the sequence-specific part of the inner primers and the entirety of the outer primers, adding 2°C for an adenine or a thymine and 4°C for a

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guanine or a cytosine, together with estimations of melting temperatures from the primer generating tool, and choosing a temperature that was believed to be sufficiently low to allow all primers to anneal successfully.

The success of PCR reactions were evaluated by running aliquots of the reaction mixture on 1% agarose gels, pre-stained with GelRed (Biotium). In the case of multiplex reactions, singleplex reaction mixtures for the primers participant in the multiplex were prepared and dilutions of the multiplex reaction mixtures were used as template for the singleplexes. The product of these singleplexes were then checked on gels, on the assumption that if and only if the multiplex had been successful would the singleplex be successful in regards to that specific primer.

Results

Primer Layout and Design

The primers required for the project were subject to a number of criteria set by the intended sequencing platform, the parameters of adjacent primers, as well as the nature of the mtDNA itself.

The external criteria set by the Illumina Paired End sequencing on the MiSeq is stated as a maximum of 550 bases per primer-amplified segment, including the primer sequences, for sufficient coverage of the entire segment. This includes an overlap of 50 bp at the centre for better coverage of the ends of the reads. This is due to the fact that, as an ensemble

sequencing-by-synthesis (SBS) method, the read length when sequencing on the MiSeq is limited by the reliability of the synchronous incorporation of the correct base to each strand in the cluster. In each step of the sequencing, the correct base has to be incorporated exactly once and be measured accurately, followed by the removal of the extension-blocking agent, allowing the next base to be incorporated and measured. As the sequencing proceeds, errors are eventually introduced, wherein bases fail to be properly incorporated in certain strands, leading to portions of the cluster lagging behind the others, giving 'false' signals. As these errors accumulate, the signal-to-noise ratio will decrease, ultimately to the point where bases can no longer be accurately detected. The number of bases into the sequencing where this threshold is reached dictates the read length of the method in question. [12]

It is, however, possible to use segments of sizes approaching 600 bp by utilising the Illumina

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stitching algorithm to combine an overlap of at least 10 bp to a single read, using consensus and quality data from the two reads, allowing the use of larger inserts. The upper limit for the size of a DNA insert, including primer sequences, was thus set to 590 bp. Subtracting the length of the primer sequences from the inserts leaves approximately 550 bp sequenced in each insert, as primers are ideally around 20 bp long. With this average fragment length, it was estimated that 32 fragments would be needed in order to fully cover the 16727 bp reference genome, with a reasonable margin for overlaps and difficult-to-align stretches of DNA. [13]

Sequencing 32 individual 550 bp long sequences would yield a total of 17600 bp, leaving a margin of 837 bases when compared to the 16727 bp of the reference genome. Spread out over 32 fragments, this enables a variance of 27 bp per fragment, providing a certain degree of freedom when aligning the primers. Finally, in order to fully cover the mitochondrial genome, the fragments cannot average lower than 523 bp (563 bp with the primers included).

32 primer pairs is also a desirable number from a practical design point of view, as sets of 32 fit evenly on 96-well plates as well as in multiples of eight, corresponding to the width of common laboratory equipment.

These 32 primer pairs must then be laid out in an interconnecting fashion, where each forward primer must be placed slightly upstream of the reverse primer of the previous pair, relative to the leading strand, so that every base is sequenced independently.

Figure 4: Schematic representation of the overlapping orientation of primers, highlighting how all parts of the template are covered by amplified fragments in an interlocking fashion. Template DNA represented by the wide yellow line, the primers by red and orange arrows (alternating colours purely for visual clarity) and the amplified fragments in corresponding colours below the template.

Another limiting factor for the placement of the primers is the repeating region of the mtDNA inside which the primers cannot reliably be placed. This is due to the fact that the repeating region, as indicated by its name, is comprised of multiple repetitions of the same DNA motif, meaning that a primer that is complementary to a site in this region is thus complimentary to a

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large number of sites, upstream and downstream of the intended annealing site, at every place where this motif repeats itself. In the domestic canine mtDNA, this repeating region alternates between two almost identical 10 bp segments, only differing in one position. This region covers bases 16131 through 16430 of the reference genome, but can vary greatly in size between individuals due to differing numbers of repeats of the two 10 bp motifs. [14]

The option of not including the repeating region was considered, as the size differences may mean that longer repeating regions would not be completely sequenced by the Illumina Paired End sequencing method, and shorter ones would be sequenced to redundancy, but possibly without the means to tell to what extent, represented in figure 5.

Figure 5: Schematic representation of the different possible results when sequencing the repeating region. Due to its varying size between individuals, coverage will vary, and due to its repeating nature, conclusive

alignments cannot be guaranteed.

It was decided to attempt to sequence the repeat region to the highest extent possible, as full coverage of the rest of the mtDNA appeared to be achievable with the remaining 31 primer pairs, meaning that no information would be lost from trying to sequence the repeat region as well. Including the repeat region as an amplified segment would also ensure that the bases immediately preceding and following it would actually be included in the sequencing, something that could otherwise not be achieved, as primers cannot reliably be aligned inside the repeat region.

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With this in mind, the primers were aligned, starting from the primer pair upstream of the repeating region, placing the reverse primer as close to the repeating region as possible, followed by the pair covering the repeating region, ensuring enough room after the repeating region to align the forward primer of the next primer pair.

The primers were designed using the NCBI Primer BLAST tool [15]. The Canis familiaris reference mitochondrion genome entry [13] was used as the template to which the primers were to be aligned. The PCR product size was set to a maximum of 590 bases and a minimum of 540 bases, to ensure coverage of the whole mtDNA sequence. Remaining parameters were subject to dynamic modifications depending on the ease or, rather, difficulty with which primers could be aligned. TM was desired to be between 52 and 60 degrees Celsius, with an optimal temperature of 56 degrees. The allowed difference in TM between the primers in a pair was initially set at 3 degrees, but was subject to increases in cases where primers could otherwise not be aligned.

The initial advanced settings were for a primer size between 17 and 23 bases with 20 as an optimum, a GC-clamp of 2, maximum poly-X sequences of 4, and maximum 3’ GC content of 3. GC content was desired to be between 40 and 60% and due to the nature of the

mitochondrial DNA, ‘Avoid low complexity regions for primer selection’ was unchecked.

Primers were then generated by specifying a stretch of approximately 50 bases within which the forward primer was allowed to align. The starting point of the first stretch was dictated by the end of the repeating region, while all subsequent alignment areas were instead dictated by the location of the reverse primer in the previous primer pair, i.e. in relation to the leading strand, each forward primer had to end before the reverse primer of the preceding pair

‘started’.

Due to the structure of the mtDNA and the rigidity of where the next primers had to be aligned, in relation to the preceding pairs, it was often difficult to align primers according to the above-mentioned ‘optimal’ parameters, which necessitated that the conditions were made less stringent, on a primer-by-primer basis. Initially, the stretch of bases allotted to the

alignment of the forward primer would be extended, in the hopes of finding a primer without having to lower the other requirements placed on the primer. Failing this, as moving the primer too far back would in the end compromise the possibility of covering the entire mtDNA in the chosen number of primers, the remaining parameters were in turn made less stringent. The decision on what parameter to change was aided by the error message given by

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for the failure, e.g. TM difference too high, too long poly-X sequence, or lack of GC clamp.

Decisions were also made by observing the surrounding sequence manually, and thereby decide whether or not certain changes were appropriate. For each primer pair, the changes to the parameters that were deemed to cause the least impactful changes to the overall structure of the primers were chosen.

To limit the use of template DNA, which is available in limited amounts, primers that would amplify longer parts of the mtDNA were required. These would then be used as templates for the aforementioned 32 primer pairs, also creating a sort of nested PCR [26], which reduces the likelihood of generating unspecific PCR products. It also serves the purpose of generating template that is in solution, not bound to FTA cards.

The 32 primers pairs will from here on be referred to as ‘Inner Primers’ and these new, analogously dubbed ‘Outer Primers’ were aligned in much the same manner as the inner primers, interlocking with each other, but also taking care not to overlap with the alignment sequences of the inner primers.

Figure 6: Schematic representation of the interlocking design of the outer primers. Template DNA represented by the wide yellow line, the primers by blue and green arrows (alternating colours purely for visual clarity) and the amplified fragments in corresponding colours below the template.

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Figure 7: Schematic representation of how the outer primers fully cover a set of four inner primers. Template DNA represented by the wide yellow line, the inner primers by red and orange arrows and outer primers by blue and green arrows (alternating colours purely for visual clarity) and the amplified fragments in corresponding colours below the template.

The outer primers were designed to cover four inner primers each, resulting in eight outer primer pairs, each amplifying around 2200 bp long sequences. These were to serve as both a way of amplifying the original templates, which is available in limited amounts, and as a way to create a nested PCR, reducing the complexity in subsequent PCR reactions.

As detailed previously, in order to utilise the Illumina Dual-Indexed Paired End sequencing protocol, a number of additional specific sequences need to be present in the primers. The basic Illumina sequencing relies on random fragmentation of sample DNA, followed by ligation of specific adaptors to the fragments, which enable bridge amplification of the fragments on the sample slide, as well as containing the primer alignment sequence for the sequencing-by-synthesis steps.

As this project endeavours to sequence the entire mtDNA of thousands of individuals in specific, predetermined PCR-amplified segments, this random fragmentation approach to creating to DNA inserts to which adaptors are ligated is not appropriate, as it would require separate libraries for each individual and involves increased labour and cost, as well as removing the specificity of using primers to ensure full coverage. Instead, the Read Primer parts of the adaptor sequences are added single-strandedly to the 5’ end of the forward and reverse inner primers as handles, making these increase in size significantly. In order to complete the sequencing-enabling structures for Dual-Indexed Paired End Sequencing, an additional PCR step is required. This step will be used to introduce the outermost adaptor sequences that allow ligation to the slides, P5 and P7, as well as the two index sequences, i5 and i7.

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Figure 8: Schematic overview of the two PCR steps that complete the sequencing construct. The top step uses specific inner primers (shown in dark grey) with attached partial adaptor sequences containing read primer complementary sequences (shown in yellow and light blue). The second step adds the outer adaptor sequences (shown in red and dark blue) and the indices (shown in light and dark green) by completing the previously added adaptors.

The final construct, shown above in figure 8, consists, from left to right, of the P5 flow cell attachment sequence, the i5 index barcode, the Read 1 Primer complementary region, the forward insert specific primer, the DNA insert, the reverse insert specific primer, the i7 index complementary region (which doubles as the Read 2 complementary region when read in the other direction), the i7 index barcode, and the P7 flow cell attachment sequence. The

difference in structure between the default ligated adaptor and this PCR-generated construct lies in the forward and reverse insert specific primers, which enable the sequencing of

specific, predetermined parts of the sample DNA, but from a sequencing stand point, these are merely treated as a part of the DNA insert, and do not influence the sequencing itself in any way.

Using 32 inner fragments per individual, and sequencing 1152 individuals in parallel, given the total read output of the MiSeq v3 sequencing kit [27], an equal distribution of reads between the fragments would, in an ideal situation, provide a redundancy of 600 reads per fragment and individual.

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PCR Reactions, Viability of Primers and Multiplex Set Ups

Initially, the first eight inner primers, from Eurofins, were tried in singleplex, 0.1 µl TagTaq, 30 cycles.

Figure 9: First attempt at amplifying the first 8 inner primers (0.1 µl TagTaq, 30 cycles) flanked by two DNA ladders (Low Range, 3% TopVisionAgarose #RO491 25-700 bp). Ladders are smeary and dissimilar, and exposure is high in an attempt to visualize potential product.

Bands were very weak and smeary. As this was true for the ladders too, as well as for other gels run by others in the lab at the same point in time, part of the fault may, in this case, lie in the gel bath itself.

Next, the same primers were tried again, resulting in a gel with much sharper bands but still very weak product bands, showing only for primers 1-3 (Figure 10), but submitting the remaining reaction mixtures to a subsequent extra 12 cycles showed more clear results (Figure 11). The gel after the additional 12 cycles shows bands of the expected size for primers 1-3 multiple, as well as multiple bands of lower sizes, presumed to be various primer dimers. These results prompted the decision to increase the number of cycles for the inner primers to 40. Low processivity of the enzyme was suspected.

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Figure 10: Second attempt at amplifying the first 8 inner primers (0.1 µl TagTaq, 30 cycles), flanked by two DNA ladders (Low Range, 3% TopVisionAgarose #RO491 25-700 bp, and M, 1% TopVision LE GQ Agarose

#RO491 250-10000 bp). Ladders are clearer but product is very weak, faintly visible for primers 1 through 3.

Figure 11: Second attempt on first 8 inner primers after 12 additional PCR cycles. Primers 1 through 3 are clearly visible. Samples flanked by two DNA ladders as before (Low Range, 3% TopVisionAgarose #RO491 25- 700 bp, and M, 1% TopVision LE GQ Agarose #RO491 250-10000 bp). The two rightmost lanes before the M ladder are primers 1 and 2 from a different sample compared to the first eight lanes.

Reactions for the same eight primers were then run using twice the amount of enzyme, 0.2 µl, for 40 cycles, and 10 times as much enzyme, 1 µl, but remaining at 30 cycles. The 0.2 µl, 40 cycle run showed product of the expected size for all primers except primer 8, while the 1.0 µl, 30 cycle run did not yield any product. Whether the latter was caused by a laboratory mistake or a result of imbalances between the reaction components due to the increase in enzyme concentration, or simply still too few amplification cycles was not further investigated.

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Figure 12: Inner primers 1 through 8 amplified with 0.2 µl TagTag and 40 cycles, in duplicate, M ladder. All primers except primer 8 appear clearly.

Figure 13: Inner primers 1 through 8 amplified with 1.0 µl TagTag and 30 cycles, in duplicate, M ladder. No primers visible, possibly due to human error.

Deciding to proceed with 40 cycles and 0.2 µl enzyme per reaction for inner primers in singleplex, different annealing temperatures were investigated. Both 49°C and 52°C were tried, using the now established parameters, both yielding product for all primers apart from primer 8.

Figure 14: Inner primers 1 through 8 amplified with 0.2 µl TagTag and 40 cycles, 49°C annealing temperature to the left and 52°C annealing temperature to the right, Low Range ladder. All primers except primer 8 appear

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Inner primers were also tried in multiplex, initially in quadruplexes of primers 1-4 and 5-8, 50 cycles, yielding vague primer dimer products. At the same time, the eight outer primers were run for the first time, using TagTaq, 50°C annealing temperature and 40 cycles, but no product was obtained. Figure 15 below shows these results, using inner primer 1 as a positive control.

Figure 15: Attempt at amplifying the 8 outer primers using 0.2 µl TagTaq, 50°C annealing temperature and 40 cycles, in duplicate, with inner primer 1 as positive control, M ladder. The two rightmost lanes contain inner primer multiplex attempts, primers 1-4 and 5-8, 50 cycles. No outer primer product visible, and only unspecific product visible for the inner primer multiplexes.

The outer primers and the multiplex attempts were retried using both 5 minutes and 10 minutes extension time for both, again failing to result in the desired products.

Figure 16: Attempt at amplifying the 8 outer primers using 0.2 µl TagTaq, 50°C annealing temperature and 40 cycles, using 5 minutes extension time (left) and 10 minutes (right), with inner primer 1 as positive control, M ladder. The 4 rightmost lanes contain inner primer multiplex attempts, primers 1-4 and 5-8, 50 cycles, in duplicate. Again, no outer primer product visible, and only unspecific product visible for the inner primer multiplexes.

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In order to rule out human error, all outer primers were re-suspended from stock solution and the PCR reactions were re-run at 40 cycles and 10 minutes extension time. As yet again no product was obtained, it was suspected that TagTaq lacked the processivity required to adequately amplify the longer outer primer fragments, and PlatinumTaq was tried instead, using 45 cycles and 10 minutes extension time. This set up yielded clear product for all eight outer primers. It was subsequently concluded that TagTaq did indeed lack the necessary processivity to reliably produce the longer, outer primer fragments, and PlatinumTaq was employed for all outer primer reactions from this point onwards.

Figure 17: Outer primers amplified with PlatinumTaq, 45 cycles, 10 minutes extension time, inner primer one as positive control, M ladder. All outer primers visible.

Subsequently, quadruplexes of the outer primers were set up, 1-4 and 5-8, using 0.2 µl PlatinumTaq, 45 cycles, and 10 minutes extension time, and singleplexes of each of the eight primers were run using 1µl 1:500 dilutions from the corresponding quadruplexes as template and were run for 15 cycles. These secondary singleplexes yielded product in outer primers 3, 4, 5, and 8.

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In order to further investigate the possibility of quadruplexing the outer primers, quadruplexes comprised of odd- and even-numbered outer primers, as well as a combination of outer

primers 1, 2, 6, and 7 and 3, 4, 5, and 8. As before, these multiplexes were verified by singleplex reactions based on the multiplex product, run on gels. The former combinations showed clear product for outer primer 3, 4, 5, and weak bands for 7 and 8. The latter was similar, and showed primers 3, 5, and 8 relatively clearly, and 4 weakly.

Figure 19: Singleplexes of outer primers, from 1 µl 1:500 dilutions of Even and Odd combination (i.e. 1-3-5-7 and 2-4-6-8) quadruplex template, 15 cycles, M ladder. Outer primers 3, 4, and 5 clearly visible, primer 7 and 8 vary faintly, and 1, 2, and 6 seemingly not amplified.

Figure20: Singleplexes of outer primers, from 1 µl 1:500 dilutions of 1-2-6-7 and 3-4-5-8 quadruplex template, 15 cycles, M ladder. Primers 3, 5, and 8 were visible, and primer 4 was faintly visible.

These results led to the decision to try the outer primers in duplexes, one set up with primers

Figure 18 Singleplexes of outer primers, from 1 µl 1:500 dilutions of 1-4 and 5-8 quadruplex template, 15 cycles, M ladder. Outer primers 3, 4, 5, and 8 clearly visible, 1, 2, 6, and 7 seemingly not amplified.

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1+2, 3+4, 5+6, and 7+8, and one set up with primers 1+3, 2+4, 5+7, and 6+8. The duplex reaction mixture was then used as templates for the corresponding singleplexes, using 1 µl 1:20 dilutions and run for 15 cycles. These set ups consistently yielded product for primers 3, 4, 5, and 8, similarly to the earlier quadruplexes, but primers 1 and 2 showed weak

amplification when paired together, as did primer 7 when paired with primer 5.

Figure 21: Singleplexes of outer primers from duplex set-ups (1+2, 3+4, 5+6, and 7+8), M ladder (one blank lane between the ladder and the first primer). Primers 1 through 5, and primer 8 visible, primers 3 through 5 more strongly.

Figure 22: Singleplexes of outer primers from duplex set-ups (1+3, 2+4, 5+7, and 6+8), M ladder (one blank lane between the ladder and the first primer). Primers 3 through 5, and primers 7 and 8 visible, primers 3, 5, and 8more strongly, primer 7 very faint.

Outer primers 5 and 6, one that had consistently worked and one that did not appear to work, were subsequently chosen for testing other parameters of the PCR. Singleplexes of primer 5 and 6 were run at 20 cycles and at 30 cycles, with 5 minutes and 10 minutes of extension time, i.e. four different PCR set ups for each primer. A duplex of outer primers 5 and 6 were also run at the same parameters. The gels showed 20 cycles to be too few to properly amplify the segments, and the longer extension time appeared to increase yield. At 10 minutes

extension time, outer primer 5 amplified to a higher extent than primer 6. The singleplexes performed from the duplexes showed amplification of only primer 5.

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Figure 23: Parameter tests for outer primers, using outer primers 5 and 6. From left to right, 20 cycles with 5 minutes extension time, 20 cycles with 10 minutes extension time, 30 cycles with 5 minutes extension time, and 30 minutes with 10 minutes extension time for both primer 5 and 6. 20 cycles did not yield product at any extension time, and the higher extension time yielded higher degrees of product, especially for primer 5.

Figure 24: Singleplex amplification of outer primers 5 and 6 from duplex template (imaged cropped from larger gel with other samples on). Only showing result of 30 cycle runs, ostensibly only yielding product for outer primer 5.

After the initial attempts at running the first eight inner primers, the remaining 24 inner primers were ordered. Due to the issues with getting inner primer 8 to yield product, and based on advice regarding primer purchase (personal communication with Afshin Ahmadian, Associate Professor, School of Biotechnology, Royal Institute of Technology, KTH) the new primers were ordered from Biolegio. Primer 8 was redesigned, and both the new and old version was ordered, along with primer 1, for comparison to the Eurofins primers, together with inner primers 9-32.

Firstly, primer 1 from both Eurofins and Biolegio were run in triplicate, as well as both the original and the new version of Primer 8, both from Biolegio and also in triplicate. The reactions were run as before, at 46°C annealing temperature and for 40 cycles. The results

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showed comparable results for both versions of inner primer 1 and indicate product from the re-synthesis of the original inner primer 8, but not from the new version.

Figure 25: Comparison of inner primer 1 from Eurofins and from Biolegio, and of the old and new design of inner primer 8, both from Biolegio, in triplicate. Primer 1 worked comparably well from both manufacturers, and the old design of primer 8 from Biolegio appeared to work, while the new design did not.

Next, inner primers 9 through 32 were tested in duplicate, according to the same parameters.

Due to the unexpected result from the two inner primer 8, these were also re-run and are included on the gel showing inner primers 25 through 32. The re-run did support the previous evidence in showing that the re-synthesis of the original primer 8 worked, while the redesign did not. The majority of inner primers 9-32 showed product, and the ones that did not or appeared only weakly were re-run.

Figure 26: Singleplexes of inner primers 9-16, in duplicate (one set after the other, with one empty lane in- between the sets), M ladder.

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Figure 27: Singleplexes of inner primers 17-24, in duplicate (one set after the other, with one empty lane in- between the sets), M ladder.

Figure 28: Singleplexes of inner primers 25-32, in duplicate (one set after the other), M ladder. Additionally, to the left of the ladder, the old and new design of inner 8, again showing product from the old design.

The primers to be re-run in duplicate were 9, 16, 19, 20, 22, 23, 24 and 25. The results obtained were largely inconclusive, being inconsistent between duplicates, at best showing fairly weak bands, at worst appearing almost fully blank, and overall showing a lot of unspecific product.

Figure 29: Singleplexes in duplicate of the inner primers between 9 and 32 that did not appear to yield product in the initial singleplexes. From left to right, in pairs, 9 16, 19, 20, 22, 23, 24, and 25, M ladder.

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Next, the viability of TagTaq compared to PlatinumTaq for the amplification of inner primers was assessed, at the same time investigating how well the inner primers amplify from a previously amplified outer primer segment, by setting up singleplexes of inner primers 9-12 using template from singleplex amplification of outer primer 3. The outer PCR was run at 50°C annealing temperature, 30 cycles, and 5 minutes extension time. 1 µl 1:20 dilution of the outer primer product was used as template for the inner singleplexes. These were run at 46°C annealing temperature, 40 cycles. All steps were performed in duplicate, i.e. two singleplex reactions of outer primer 3 were used for duplicates of both the TagTaq and the PlatinumTaq, totalling four singleplexes of each inner primer. All singleplexes were successful, with PlatinumTaq showing much more strongly, and the two sets of TagTag clearly differing in intensity.

Figure 30: Duplicate sets of inner primers 9 through 12 in singleplex from previous amplification of outer primer 3, comparing TagTaq (left) to PlatinumTaq (right), M ladder. The PlatinumTaq amplified inner primers show more strongly, and there is a marked difference between the two TagTaq sets, despite having been amplified under the same conditions.

Similarly, quadruplexes of inner primers 9-12, one using TagTaq and one using PlatinumTaq, were set up, still using the amplified outer primer 3, 1:20 dilution, as template. Reactions were run at 46°C annealing temperature, 30 cycles. Secondary singleplexes for verification were performed with Platinum for all reactions, on 1:20 dilutions of the quadruplex mixtures.

Results were similar between the two enzymes; inner primers 9, 11, and 12 were successfully amplified, while primer 10 was very weak.

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Figure 31: Singleplexes from quadruplexes of inner primers 9 through 12. All singleplex reactions were performed with PlatinumTaq, while one quadruplex was performed TagTaq (left) and one with PlatinumTaq (right), M ladder.

Due to the apparent failure of certain outer primers in quadruplex reactions, outer primers were re-suspended from stock and run in singleplex, as before, showing primers 3 through 8 clearly, primer 2 was weaker and primer 1 was very faint. This was to investigate degradation of the primers due to freeze-thaw cycles as the cause of the amplification failure.

Figure 32: Outer primers in singleplex, re-suspended from stock, M ladder.

Diluting all outer primer product (apart from primer 1, being significantly weaker) at a ratio of 1:20, singleplexes of all inner primers were run from their corresponding outer primer, for 40 cycles, with 46°C annealing temperature. Results showed amplification of inner primers corresponding to each of the outer primers, but not from all inner primers, even from inner primers that had previously been successfully amplified.

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Figure 33: Singleplexes of inner primers 1 through 16 from outer primer singleplexes 1 through 4, M ladder.

Figure 34: Singleplexes of inner primers 17 through 32 from outer primer singleplexes 5 through 8, M ladder.

The resuspended outer primers were then tried in quadruplex; outer primers 1, 3, 5, and 7 in one quadruplex and outer primer 2, 4, 6, and 8 in the other. They were run in duplicates of both 20 and 30 cycles, all using 50°C annealing temperature and 5 minutes. 1:20 dilutions of the quadruplex reaction mixtures were used for singleplex verification and were run for 15 cycles. The gels showed successful amplification of primers 3, 4, 5, and 8 in both the 20 cycle and the 30 cycle quadruplexes, and in the latter, outer primer 7 was also visible.

Figure 35: Duplicates of outer primer singleplexes from outer primer quadruplexes (1+3+5+7 and 2+4+6+8, i.e. Even and Odd), M ladder. Quadruplex run for 20 cycles, primers 3, 4, 5, and 8 faintly visible.

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Figure 36: Duplicates of outer primer singleplexes from outer primer quadruplexes (1+3+5+7 and 2+4+6+8, i.e. Even and Odd), M ladder. Quadruplex run for 30 cycles, primers 3, 4, 5, 7 and 8 visible, number 7 more faintly.

It was then decided to attempt singleplex inner primer amplification, using the 30 cycle outer primer quadruplex reaction mixture as template, at 1:100 dilution. The singleplexes were run for 40 cycles. Although product was only expected for inner primers corresponding to outer primers 3, 4, 5, 7, and 8, gels showed amplification of inner primers from all outer primers, including those that appeared not to have worked in quadruplex. Only two inner primers appeared to not have yielded product.

Figure 37: Singleplexes of inner primers 1 through 16, from outer primer quadruplex template (even and odd outer primer combinations), M ladder. Most inner primers visible, irrespective of whether or not the

corresponding outer primer appeared to have yielded product.

Figure 38: Singleplexes of inner primers 17 through 32, from outer primer quadruplex template (even and odd outer primer combinations), M ladder. Most inner primers visible, irrespective of whether or not the

corresponding outer primer appeared to have yielded product.

To verify this unexpected result, the entire experiment was run again, starting from the quadruplex of the outer primers, with similar results.

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Figure 39: Singleplexes of inner primers 1 through 16, from re-run of outer primer quadruplex template (even and odd outer primer combinations), M ladder. Again, almost all inner primers are clearly visible.

Figure 40: Singleplexes of inner primers 17 through 32, from re-run of outer primer quadruplex template (even and odd outer primer combinations), M ladder. Again, almost all inner primers are clearly visible.

Barcoding PCR

Following these results, the decision was made to proceed towards sequencing. For two different DNA samples, PCR1 was run in quadruplexes of odd and even numbered outer primers, 40 cycles, and PCR2 was run in quadruplexes, duplexes and singleplexes according to the pattern in figure 41, each for 25 cycles. A further four DNA samples were prepared in the same manner, but for these, PCR2 was only run on quadruplexes.

Figure 41: Duplex and quadruplex set-ups for all 32 inner primers, with corresponding names (Q1-8 for the

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Product from PCR2 were pooled together, per individual and multiplexing set-up, creating 32- plexes of inner fragments, and a 1:100 dilution of these pools were used as template for the barcode-introducing PCR3. This reaction was run for 15 cycles, at 58 °C annealing

temperature, and 5 minutes extension time. Both TagTaq and PlatinumTaq were employed, according to Table 1 below.

 

Samples   Singleplex  

TagTaq  

Singleplex   PlatinumTaq  

Duplex   TagTaq  

Duplex   PlatinumTaq  

Quadruplex   TagTaq  

Quadruplex   PlatinumTaq  

IR119   X   X   X   X   X   X  

IR126   X   X   X   X   X   X  

IR85   -­‐   -­‐   -­‐   -­‐   X   -­‐  

IR92   -­‐   -­‐   -­‐   -­‐   X   -­‐  

IR114   -­‐   -­‐   -­‐   -­‐   X   -­‐  

IR127   -­‐   -­‐   -­‐   -­‐   X   -­‐  

Table 1: Table showing the different combinations of samples, enzymes, and multiplexing variants used in PCR3 for the introduction of the barcode indices.

Concentration Measurements and Cleaning

Concentration measurements were performed on all 32-plexes after PCR3, using the Qubit dsDNA HS Assay Kit (Invitrogen, Life Technologies), followed by a cleaning step on an MBS machine (Magnetic Bead Separation) in order to remove smaller fragments than those meant for sequencing, such as loose primers and primer dimer constructs. This first

concentration measurement was performed both as a quick way of verifying product from PCR3, and as a means of estimating the relative concentration of actual product when

compared to the clean samples. The Illumina CA Purification protocol [15] was used, diluting 20 µl of PCR3 product to 50 µl using elution buffer (EB). A concentration of 14% PEG was used as precipitation buffer in order to achieve an appropriate size cut-off [17] [18]. The parameters entered into the Magnatrix OS were 50 µl sample volume, 20 µl magnetic beads, 100 µl Precipitation Buffer, 25 µl EB, and 10 minutes binding time, resulting in input volumes of 50 µl sample, 95 µl EB, 125 µl 14% PEG, 220 µl 80% EtOH, and 25 µl beads.

After MBS cleaning, a second Qubit concentration measurement was performed on all samples, in order to estimate the actual product concentration, from which the pooling of samples for the sequencing was subsequently based. The samples were also run on

BioAnalyzer (Agilent Technologies, 1000 kit) for a visual verification of the success of the

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cleaning step. The desired products are expected to be in the range of 600-700 bp, due to the base fragment being around 550 bp, to which large additional adaptor sequences have been added.

Figure 42: Bioanalyzer results of all samples apart from the singleplex PlatinumTaq set ups, which were on a separate Bioanalyzer run. All samples are successfully cleaned, showing no short, unspecific products, and those run with PlatinumTaq clearly showing a peak at the expected size range of 600-700, while peaks are very small for those run with TagTaq.

Both assays indicated higher yields of specific product from the sample set-ups run with PlatinumTaq than from those that were performed with TagTaq, and product above the detection cut-off for all samples except one (Table 2). In the case of the four additional individual samples, these were pooled into one sample prior to the second cleaning step.

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Sample  

Concentration  before  cleaning   Concentration  after  cleaning   In  assay  

[ng/ml]  

In  sample   [µg/ml]  

In  assay   [ng/ml]  

In  sample   [µg/ml]  

IR119ST   20.6   4.12   1.32   0.263  

IR119SP   50.2   10.0   23.9   4.78  

IR119DT   21.0   4.20   1.06   0.212  

IR119DP   70.8   14.2   25.6   5.12  

IR119QT   14.7   2.94   <0.5*   -­‐  

IR119QP   48.7   9.74   21.6   4.33  

IR126ST   20.0   4.0   1.61   0.322  

IR126SP   36.3   7.27   22.3   4.45  

IR126DT   18.8   3.77   1.24   0.248  

IR126DP   35.6   7.12   19.9   3.99  

IR126QT   23.2   4.64   1.42   0.284  

IR126QP   37.6   7.51   18.0   3.59  

IR85QT   17.6   3.52  

1.15   0.230  

IR92QT   11.6   2.33  

IR114QT   14.5   2.89  

IR127QT   22.7   4.53  

Table 2: Overview of the amount of PCR in the different samples before and after cleaning using the Illumina CA Purification Protocol on the MBS [17].

Initial Sequencing

All 32-plexes were then pooled for sequencing on the MiSeq, using the MiSeq Reagent Kit V2, 300 cycles (Illumina), i.e. paired-end sequencing of 150 bases from each end. After demultiplexing, the results, shown in abbreviation in Tables 3 and 4, were analysed, and table 5 provides a colour coding key, used to highlight the different magnitudes of reads. Results showed generally lower numbers of reads than anticipated, and while not being completely conclusive nevertheless showed clear trends in successful amplification and sequencing. The main implications were that sample set-ups where PlatinumTaq had been used for PCR3 had overall generated larger numbers of reads than those that had been performed with TagTaq, and that the inner fragments corresponding to outer fragments 1, 2, and 6 (inner fragments 1 through 8, and 21 through 24) had yielded far fewer reads than the remaining ones. The latter trend was particularly noticeable among the first 8 inner fragments, with only two out of 16 deviating from the pattern, while the pattern for fragments 21 through 24 was not as

pervasive.

References

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