Faculty of Science
METAGENOMIC APPROACH FOR UNDERSTANDING PERMAFROST MICROBIAL
COMMUNITY FUNCTIONAL GENE COMPOSITION
2024 | CADE GLEN TORJUSSON

B.Sc. HONOURS THESIS – CHEMICAL BIOLOGY

METAGENOMIC APPROACH FOR UNDERSTANDING PERMAFROST MICROBIAL
COMMUNITY FUNCTIONAL GENE COMPOSITION

by

Cade Glen Torjusson

A THESIS SUBMITTED IN PARTIAL
FUFILLIMENT OF THE REQUIREMENTS FOR
THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
In the
DEPARTMENT OF BIOLOGICAL AND
PHYSICAL SCIENCES
(Chemical Biology)
This thesis has been accepted as conforming to the required standards
by: Eric Bottos (Ph.D.), Thesis Supervisor, Dept. Biological Sciences
Kingsley Donkor (Ph.D.), Co-supervisor, Dept. Physical Sciences
Natasha Ramroop Singh (Ph.D.), External Examiner, Dept. Biological Sciences

Dated this 17th day of April 2024, in Kamloops, British Columbia, Canada

ABSTRACT

This study aimed to create and validate metagenomic libraries for sequencing to establish
baseline understanding of active layer and permafrost microbial community functional gene
composition and observe the changes that occur in functional gene abundance in these microbial
communities when permafrost soils thaw. The soils used in this study were obtained from
Cambridge Bay, Nunavut in the Canadian High Arctic. Five different thaw treatments, differing
in soil compositions of permafrost and active layer soil were prepared in triplicate and incubated
at 8 °C for 12 weeks. These treatments consist of permafrost and active layer soil incubated on
their own and three other treatments with varying mixtures of both soils, designed to test how
they will respond to permafrost thaw. Microbial communities were expected to change uniquely
in their respective treatments over the experimental period. DNA and RNA extractions were
performed on pre-thaw samples and samples after 12 weeks of incubation. Obtained DNA
concentrations ranged from 93.5-425ng/2g of soil and extracted RNA samples were too low to
pursue sequencing. DNA libraries were successfully prepared for Illumina sequencing (as
assessed by size distribution analysis, confirmed by gel electrophoresis and Bioanalyzer assays),
library quantification using a Qubit fluorometric assay, and qPCR analysis. Size distribution
ranged from 310-570bp, and library concentrations were found to range from 0.020-53.46nM.
Metagenomic sequencing of these libraries will allow insight into the functional gene
abundance and microbial community changes that occur as these soils thaw.
Primary Thesis Supervisor: Eric Bottos
Secondary Thesis Supervisor: Kingsley Donkor

ii

ACKNOWLEDGEMENTS
I would like to thank Eric Bottos for both guiding and funding me through this research so I could
have this opportunity. Thank you to Kingsley Donkor as well for assisting in supervising and
guiding me through this project and Natasha Ramroop Singh for acting as a committee member
for my Honors defense. A final thanks to TRU and its support and assistance for undergraduate
research opportunities such as this.

iii

TABLE OF CONTENTS
ABSTRACT .................................................................................................................................... ii
ACKNOWLEGDEMENTS ........................................................................................................... iii
LIST OF FIGURES .......................................................................................................................... v
LIST OF TABLES .......................................................................................................................... vi
INTRODUCTION ............................................................................................................................ 1
Permafrost Background and Classification ............................................................................. 1
Permafrost Organic Matter and Thawing Permafrost Effects ................................................. 2
Selective Pressure present on Bacteria Present in Permafrost ................................................. 3
Principles of Metagenomics .................................................................................................... 4
MATERIALS AND METHODS ...................................................................................................... 8
Treatment of Soil Samples ...................................................................................................... 8
DNA/RNA Extraction from Soil ............................................................................................. 9
DNA Shearing and Size Selection ........................................................................................... 9
DNA Library Preparation ........................................................................................................ 9
qPCR on DNA Libraries ....................................................................................................... 11
RESULTS AND DISCUSSION .................................................................................................... 12
CONCLUSIONS AND FUTURE WORK ..................................................................................... 22
LITERATURE CITED ................................................................................................................... 25
APPENDIX .................................................................................................................................... 28

iv

LIST OF FIGURES
Figure 1. Global distribution and classification of permafrost in northern and southern
hemispheres. Classification of the permafrost is displayed through varying color intensity (Brown
et al. 1997)…………………………………………………………………………………………2
Figure 2. Metagenomic assembly process, obtained genomic DNA goes through a series of sorting
and filtering process shown, to reconstruct original genomes of the microbial species………….…6
Figure 3. Visual representation of how overlapping regions of short-read sequences can allow
extension and reconstruction of original genomic regions defined as contigs…………………….7
Figure 4. Schematic representing the dA-tailing of DNA strands and their subsequent ligation to
complimentary adaptors (Figure from New England Biolabs)…………………….…………..…10
Figure 5. Well plate distribution of prepared controls, samples, and standards used in qPCR
analysis of DNA libraries………………………..………….……………………………………11
Figure 6. Agarose gel showing confirmed average size of 300bp DNA fragments from samples
191, 190, and 188……………………………………………………………………………...…15
Figure 7. Standard curve obtained from six prepared standards for qPCR analysis, showing cycle
threshold against concentration of prepared standards on a logarithmic scale………………..…..17

v

LIST OF TABLES
Table 1. DNA extraction yields from soils before thaw experiments (T0)………………………..13
Table 2. DNA extraction yields from after 12 weeks at 8˚C…………………………………..….14
Table 3. Average fragment size and respective DNA concentrations obtained from samples after
library preparation, obtained by Bioanalyzer high sensitivity DNA gel chip analysis……………16
Table 4. Interpolated concentrations of amplifiable molecules in permafrost-based DNA libraries
from qPCR analysis…………………………………………………………………………...….18
Table 5. Interpolated concentrations of amplifiable molecules in active layer-based DNA libraries
from qPCR analysis………………………………………………………………………………19
Table 6. DNA concentrations of each permafrost-based library after library preparation; obtained
using a Qubit Fluorometer……………………………………………………...……………...…21
Table 7. DNA concentrations of each active layer-based library after library preparation; obtained
using a Qubit Fluorometer………………………………………………………………………..28

vi

INTRODUCTION
Permafrost Background and Classification
Permafrost is defined as a body of soil that has been continuously frozen for a minimum of
2 years (Pokrovsky O. 2013). These conditions impart a harsh habitat for organisms, and as
such, these soils are typically inhabited by psychrophilic bacteria that can survive in this cold
environment (Margesin R, Collins T. 2019). The cold-adapted bacteria form microbial
communities that are composed of several species and subspecies that vary between locations
and with variation in environmental and nutritional conditions of the soil (Jansson K, Taş N.
2014).
Permafrost regions contain large areas of permafrost soils that are exposed on the surface or
buried beneath surface soils (Povrovsky O. 2013). Permafrost occupies an estimated 14-16 x
106 km2 and underlies 16% of Earth’s exposed land surface (Obu J. 2021). This includes roughly
15% of the exposed surface in the northern hemisphere and 100% of the underlain ice-free ground
in the Antarctic (Obu J. 2021). However, ground throughout much of Antarctica is not defined
as permafrost, as it is composed primarily of layers of ice sheets, not soil (McGee 2022). For
this reason, permafrost soils are found almost exclusively in the far north of the northern
hemisphere or in high elevation regions where the climate is cold year-round (McGee 2022).
Permafrost can be further classified based on its continuity. Continuous permafrost defines
regions where 90-100% of the underlying landscape consists of permafrost (Carpino et al. 2021).
Discontinuous permafrost defines regions where 50-90% of the underlying landscape is
permafrost. These regions of discontinuous permafrost show patchy complicated networks of
permafrost soil, influenced by external factors such as thick vegetation and geographical
features that may affect exposure and heat energy absorbed by the soil. Sporadic permafrost
defines regions where 0-50% of the underlying landscape consists of permafrost, with
distributions influenced by similar factors as discontinuous permafrost.

1

Figure 1. Global distribution and classification of permafrost in northern and southern hemispheres.
Classification of the permafrost is displayed through varying color intensity (Figure from Brown et
al. 1997)

Permafrost Organic Matter and Thawing Permafrost Effects
Permafrost is an important store of organic material, containing 25-50% of the total global
soil carbon pool (Mackelprang et al. 2016). Global estimates put this at roughly 1380-1580Pg of
organic carbon (Walter et al. 2018). Metabolic functions of microbial communities within soils
rely heavily on organic matter degradation, and metabolism of organic compounds are an
important source of materials and energy for microorganisms (Sipes K. et al. 2022).
The layer of soil overlaying the permafrost soil is referred to as the ‘active layer’. The
active layer is typically more exposed to the surface and thus is subject to the natural temperature
changes that come with seasonal changes, extreme weather, and changing global conditions. The
active layer freezes and thaws seasonally, allowing the breakdown, and cycling of organic
materials during the thawed period. The permafrost layer is in a continuous frozen state, heavily
limiting the breakdown of these organic materials resulting in the large quantities of sequestered
carbon (Sipes K. et al. 2022). The thawing of permafrost can lead to utilization of this previously
inaccessible organic material. This is an environmental concern as microbial metabolism will
2

convert large amounts of this stored carbon into methane and carbon dioxide, releasing these
greenhouse gasses into the atmosphere (Winder JC. Et al. 2023).
Predictions based on the current rate of permafrost thaw estimate that 6-33Pg of this carbon
will be released as carbon emissions from the soil by microbial metabolism by the year 2100,
resulting in a 0.3°C ± 0.2 increase of temperatures around the globe (Winder JC. Et al. 2023).
These conditions create positive feedback cycles that feed higher temperatures for more thaw, thus
resulting in more organic matter degradation.

Selective Pressures on Bacteria Present in Permafrost
The microorganisms present in the permafrost and active layers are adapted to the
conditions of their respective environments for metabolic and physiological needs. Permafrost is
a cold environment inhabited by psychrophilic bacteria, which optimally grow at temperatures
below 15°C. These bacteria have adapted to extreme cold environments through methods
mentioned previously such as membrane fluidity changes, antifreeze peptide inclusion, and
metabolic reductions, common psychrophilic bacteria contained in these permafrost soils include
acidobacteria, proteobacteria, actinobacteria, and bacteroidetes, to name a few from the typical
large masses of diversity (Monteux S. et al. 2018). Psychrotolerant bacteria, can also survive at
subzero temperatures but grow optimally at temperatures around 20-26°C (De Maayer et al. 2014).
These bacteria can survive in these cold conditions through adaptations similar to psychrophilic
bacteria, although typically to a less extreme extent respective to their environment (Monteux S.
et al 2018). Metabolic adaptations must occur as well due to the low nutritional availability. Stress
response pathways are activated, and remodeling of metabolic pathways occurs to compensate for
the low availability of organic material that can be utilized. These metabolic pathways will
typically consist of breakdown of nutrients from the soil such as sulfur metabolism and cystine or
methionine metabolism. The key change psychrophiles have differentiated in their systems is the
inclusion of anti-freeze peptides and changes in typical protein compositions to resist ice crystal
formation and cold denaturation of the proteins within the organism. Antifreeze peptide
interactions are not completely understood, although studies have shown that these peptides resist
ice formation by adhering to ice crystals disrupting water-ordering, resisting ice crystal formation
within the cell (Kuiper 2015).
3

Principles of Metagenomics
Metagenomics is an analytical approach that aims to reconstruct individual microbial
genomes from DNA extracted from diverse microbial communities (Zhang 2023). In traditional
microbiological methods, bacteria are isolated and cultured using standard techniques that allow
proliferation on artificial media, isolating the cultured microorganisms for further analysis. These
methods, however, are limited, as less than 1% of environmental bacteria can be cultured using
these approaches (Hofer 2018). Metagenomic sequencing avoids this shortcoming by allowing
DNA from a mixture of microorganisms to be analyzed in a single environmental sample,
bypassing the need for microbial isolation. Microbes in this respect can be simultaneously sampled
to reconstruct genomes of the diverse microbial populations present within an environment (Zhang
2023).
Metagenomics follows a logical progression, as shown in Figure 2. Preparation requires
extracting the genomes of interest from a sample and fragmenting them into short pieces, as most
sequencing platforms are optimized to sequence short DNA fragments (<1000 bp) (Smith 2022).
The fragmentation of these genomes can be achieved by methods such as enzymatic means or
ultrasonication, to produce random fragments. This random fragmentation allows discontinuity
between DNA fragments, assisting in genome assembly. The fragmented DNA is prepared for
sequencing by preparation of DNA libraries and addition of nucleotide barcodes for tracking. The
DNA libraries must then be sequenced and then metagenomic methods can be conducted. Once
these short fragments of DNA are sequenced, they are assembled into longer fragments classified
as ‘contigs’ (Nam et al. 2023). Contigs are constructed from the smaller fragments of DNA by
analyzing overlapping segments of genomic regions (Green 2024). Some drawbacks are associated
with short-read sequences such as constructing conserved sequences correctly amongst mixed
microbial genomes and creating high quality genome maps with low abundance genomes (Zhang
2023). The short-read sequences created will have overlapping regions with other short reads
derived from the same microbial species thus allowing contiguous extension of the respective
region of the DNA as demonstrated in Figure 3.

5

Figure 2. Metagenomic assembly process, obtained genomic DNA goes through a series of
sorting and filtering processes shown to reconstruct original genomes of the microbial species
(Figure from NGS Analysis).

Contigs are further sorted in a process known as binning. Binning sorts the contiguous
sequences and short reads obtained into groups designated as bins. These bins profile sequences
into groups deemed to be from the same or similar genomes (Wang et al. 2024). This is a key
point in metagenomic analysis, as it attempts to separate the diverse genomes back from a
complex mixture of DNA to their original respective genomes. The contig and binning processes
can make use of existing genomic databases to assist with genome reconstruction. Microbial
species composition and functional gene abundance can be interpreted from the reconstructed
genomes (Nam et al. 2023).

6

Figure 3. Visual representation of how overlapping regions of short-read sequences can allow
extension and reconstruction of original genomic regions defined as contigs (Figure from
National Human Genome Research Institute).

Objective
This research aims to create metagenomic libraries from the bacterial communities present in
active layer and permafrost soils. This will allow further metagenomic research to understand
and classify the bacteria present in both the permafrost and active layer with respect to their
functional gene composition, and their response to thaw over time. The bacterial communities
within their respective soil layers are very different, as they have been shaped by different
selective pressures within their environment. To recreate the permafrost soil thaw conditions
experienced with rising global temperatures, selective soil compositions were thawed and
7
incubated at 8°C for 12 weeks. Comparisons of pre-thaw and post-thaw bacterial meta-genomes

will give insight into the emergent selective pressures on these bacteria as their environments
change, and how these new environments are utilized by these bacteria based on analyses of
functional gene abundances in their respective communities.

MATERIALS AND METHODS

Treatment of Soil Samples
Soil samples were collected from Cambridge Bay, Nunavut in July-August 2023 and were stored
at -20°C until use. Soil was distributed amongst 24, 60 mL serum bottles. Soil samples were
prepared by a fellow honors student Chloe MacLean for her project, and the soils analyzed in this
study represent a subset of samples from that experiment. The 24 samples were equally distributed
among eight treatments. A treatment of three samples contained solely permafrost soil, a second
treatment of three samples contained solely active layer soil, a third treatment contained a mixture
of active layer and permafrost soil in a 1:10g ratio, a fourth treatment contained solely permafrost
soil but treated with nutrients extracted from the active layer soil, and a fifth treatment contained
solely active layer soil treated with the nutrients from the permafrost layer soil. These samples
were thawed and incubated for 12 weeks at 8°C. After 12 weeks, DNA and RNA was extracted
from the microorganisms present in the soil treatments. Time zero (T0) treatments were not
incubated prior to DNA and RNA extraction. These T0 treatments included a treatment of three
samples that contained solely permafrost soil, a second treatment of three containing solely active
layer soil, and the third treatment containing a mixture of active layer and permafrost soil in a
1:10g ratio respectively.
Water soluble nutrients for the treatments listed above were extracted from active layer or
permafrost soils to examine the influence of nutrient mixing between these two soil layers. To
extract nutrients, soil was placed in a 15 mL tube with Milli-Q water to fill to the 10 mL mark. and
left for one hour and shaken every 15 minutes. This mixture was then centrifuged at 3000 rpm for
3 minutes where 5 mL of the supernatant was extracted and placed in labeled vials for later use.

8

DNA/RNA Extraction from Soil
To sample microcosms for DNA and RNA extraction, samples were vortexed for 30 s on
high and mixed with a scoopula for 60 s to ensure homogeneity and equal bacterial distribution
among the soil. A total of 2.00 g ± 10% of soil was weighed into each Powerbead Soil tubes for
DNA/RNA extraction. The RNA extractions were performed using a RNeasy Powersoil Total RNA
Kit (Qiagen, Germany), and DNA extractions were performed using a RNeasy Powersoil DNA
Elution Kit (Qiagen, Germany). DNA extract concentrations were determined using a High
Sensitivity DNA Assay kit on a Qubit fluorometer. RNA concentrations obtained were deemed too
low for further analysis.

DNA Shearing and Size Selection
To produce DNA fragments of the appropriate size for metagenomic sequencing, 50 µL of
each DNA extract was placed in an AFA fiber microtube. Samples were ultrasonicated using a
Covaris Ultrasonicator program to shear the average base pair size to 300 bp.
From the 50 µL sonicated, 10 µL was taken from three separate samples and run on a 2%
agarose gel to confirm the resulting size distributions. The gel was run at 80 V for 45 minutes in a
1x TAE buffer solution. Gel electrophoresis was completed with 50 bp and 100 bp Biohelix DNA
ladders to validate average size produced by the shearing program.
Size validation was further confirmed on a Bioanalyzer with 1 µL of sample using a High
Sensitivity DNA Assay kit (Agilent).

DNA Library Preparation
DNA library preparation was performed using a NEBNext Ultra™ II DNA Library Prep
Kit for Illumina (New England Biolabs), following manufacturer’s protocols. The fragmented
DNA was placed in an enzyme reaction mixture to dA-tail the 3` ends of the DNA. This process
involves enzymatically adding an adenosine nucleotide on to the 3’ ends of the DNA strands to
prepare for adaptor ligation, as shown in Figure 4 (New England Biolabs). The adaptor reaction
mixture was diluted 25-fold for permafrost DNA samples and 10-fold for active layer DNA
9

samples, based on DNA concentrations obtained. The samples were then ligated with their
respective adaptors. In this process, the adaptors form hairpin loop structures at the tails of the
DNA. These hairpin loops must be excised if the DNA is to be processed for further library
preparation steps. To excise the hairpin loops, USER enzyme (New England Biolabs) contains
recognition sites with nuclease activity at the adaptor sites. USER enzyme was aliquoted into the
samples to cut

the

ends of the adaptor sequences

opening

the hairpin

loops.

Figure 4. Schematic representing the da-tailing of DNA strands and their subsequent
ligation to complimentary adaptors (Figure from New England Biolabs).

DNA was isolated from the prepared libraries using NEBNext sample Purification Beads
(New England Biolabs) with use of a magnetic rack for bead isolation. These beads contain free
carboxylic acid groups on their surface to bind and isolate the DNA from the solution (Lluis et. al
2015). The isolated DNA samples were ligated to individual barcodes to create metagenomic
libraries. These barcodes are unique, known nucleotide sequences, that allow the tracking of
individual libraries by this attached sequence. The DNA samples at this point were sorted as
independent libraries with their own unique barcodes. Forward and reverse primers were then
attached onto the ends of the DNA for subsequent PCR reactions. Thermal cycling of the libraries
was performed for DNA amplification. The libraries were isolated from the PCR mixture using

10

NEBNext sample Purification Beads. Concentrations were determined using qPCR and a Qubit
High Sensitivity dsDNA assay. Samples were then stored at -21°C.

qPCR On DNA Libraries
qPCR was completed to determine the concentration of amplifiable DNA in each library.
The procedure was conducted following the NEBNext Library Quant Kit for Illumina (New
England Biolabs). Serial dilutions of 1:1000, 1:10’000, and 1:100’000 were prepared for each
sample in NEBNext Library Quant Buffer. The dilutions were performed to bring the DNA
concentrations of the libraries to fall within the range of the standards supplied. Seven supplied
standards were used for library reference, containing concentrations of 100 pM, 10 pM, 1 pM, 0.1
pM, 0.01 pM, 0.001 pM, 0 pM respectively. Standards and diluted library samples were taken in
4µL aliquots and placed into 16 µL of PCR master mix. All standards and libraries were prepared
with 0.2 µL of ROX as a passive reference for the qPCR instrument. Prepared standards and
libraries were loaded onto a 96-well plate according to Figure 5. The qPCR assay conditions
consisted of an initial denaturation at 95°C for one minute, proceeded by 35 cycles of denaturation
at 95°C for 15 seconds and extension at 63°C for 45 seconds.

Figure 5. Well plate distribution of prepared controls, samples, and standards used in qPCR
analysis of DNA libraries.

11

RESULTS AND DISCUSSION

The purpose of the study was to prepare DNA libraries from permafrost and active layer soil
microbial communities, for further metagenomic study. DNA and RNA extractions were both
undergone however, obtained RNA concentrations were too low for analysis and functional gene
expression of the microbial communities was not analyzed. Initial soil nucleic acid extractions
were able to give an inference into the density of soil microbes present. All T0 permafrost samples
had extracted DNA concentrations below the detection limit when measured using the Qubit
fluorometer as shown in Table 1. Much lower DNA concentrations were expected in the
permafrost than the active layer as microbial abundance and activity are restricted in the
permafrost (Mohanty 2022). Increases in DNA concentrations were expected in the permafrost
soils after 12 weeks of incubation; however, DNA yields were below the limit of detection in all
but one of the incubated permafrost samples as seen in Table 2. The Qubit fluorometer used to
detect the concentrations of obtained DNA had a detection limit of <50 ng of DNA per 2 g of
soil. These results were not fully determined as having no obtained DNA in the permafrost
samples, as DNA pellets were visually noted in the extraction process. The sole permafrost
treatment showing DNA concentrations above the limit of detection was sample 45. This
treatment was a composed of permafrost soil with water soluble active layer nutrients and active
layer soil introduced. A higher abundance of microorganisms was expected in this treatment, as
the introduction of active layer soil was intended to introduce microbes from the active layer into
the permafrost sample. The active layer microbes are well adapted to thawed conditions, so
proliferation rates were expected to be greater (McDonald et al. 2023). Without metagenomic
data for confirmation, it is not certain to say that the proliferation of bacteria in this sample is
solely due to increased abundance of active layer microbes. It may be an exception as DNA
concentrations were below the limit of detection for the other samples in the treatment, although
it can be reasoned that the active layer microbes were indeed able to proliferate and take
advantage of this soil environment over the course of the 12-week period of thaw as
hypothesized. Further analysis of the bacteria present by metagenomic analysis would help to
resolve the reason for higher DNA concentrations in this sample. Earlier use of bioanalyzer
chips would also have allowed lower detection limits of the DNA libraries, assisting in preparing
in library preparation and PCR reaction
12

Table 1. DNA extraction yields from soils before thaw experiments (T0).

13

Table 2. DNA extraction yields from after 12 weeks at 8°C.

Sequencing of the DNA libraries is to be conducted by next-generation Illumina
sequencing instruments at the University of Calgary. Next-generation sequencing works best with
base pair lengths of 50-500 bp dependent upon the application (Smith 2022). Genomic DNA
obtained was fragmented into average 300 bp lengths by ultrasonication. To confirm fragment
length a 2% agarose gel electrophoresis was run with 100 bp and 50 bp DNA ladders. The agarose
gel run in Figure 6 shows confirmation of this average size. Further size confirmation was
performed using an Agilent Bioanalyzer DNA Chip. The size distribution in Table 3 displays the
DNA fragment sizes post-library preparation. Amplifiable DNA concentrations were determined
using qPCR. Six standards were used to prepare the standard curve present in Figure 7 and DNA
concentrations interpolated from this curve are present in Tables 4 and 5.

14

Figure 6. Agarose gel confirmed average size of 300 bp DNA fragments from samples 191,
190, and 188 as compared against Biohelix DNA ladders.

15

Table 3. Average fragment size and respective DNA concentrations obtained from samples
after library preparation, obtained by Bioanalyzer high sensitivity DNA gel chip analysis.

16

25.000

y = -1.498ln(x) + 12.579
R² = 0.9996

Cycle Threshold

20.000

15.000

10.000

5.000

0.000
0.001

0.01

0.1

1

10

100

Concentration (pM)

Figure 7. Standard curve obtained from six prepared standards for qPCR analysis, showing
cycle threshold against concentration of prepared standards on a logarithmic scale.

17

Table 4. Interpolated concentrations of amplifiable molecules in permafrost-based DNA
libraries from qPCR analysis.

18

Table 5. Interpolated concentrations of amplifiable molecules in active layer-based DNA
libraries from qPCR analysis.

qPCR analysis was completed to determine amplifiable DNA concentrations for the
libraries prepared. Primers used in this process amplify from the adaptor sequence ligated in
preliminary steps. Illumina sequencing operates by sequencing by synthesis from the same adaptor,
therefore, by performing qPCR from these adaptors, real time DNA concentrations are only
obtained from these amplifiable molecules that contain the ligated adaptor sequence. The
concentrations of the DNA obtained from permafrost-based samples seen in Table 4 were
significantly lower than DNA concentrations of active layer-based samples seen in Table 5.

19

PCR with a low DNA input requires precise methodology respective to DNA fragment size,
DNA input, and reagent ratios (PacBio 2022). The concentrations of DNA lying outside of a
measurable range for the permafrost samples created a challenge for preparation of library
preparation reactions. This may have led to improper ratios of reagents inhibiting proper adaptor
ligations from occurring. The concentration of the active layer-based DNA libraries obtained
from Qubit fluorometry in Table 7 are consistent with that of the active layer-based DNA
concentrations obtained from qPCR in Table 5 suggesting proper library preparation. In contrast
the DNA libraries obtained from Qubit fluorometry for the permafrost-based libraries in Table
4 are not consistent with concentrations obtained from qPCR of the permafrost-based libraries
in Table 6. This suggests that DNA is present in the prepared libraries as it is being detected
with spectrophotometric means although the adaptors are not present to amplify and detect by
qPCR methods. As previously mentioned, qPCR requires the Illumina adaptor sequences to be
present on the DNA as the primers used in this method recognize sites on the adaptor for the
PCR process to occur. The improper ligation of adaptors would therefore be reflected in the
differences in concentrations obtained through the different methods as seen.

20

Table 6. DNA concentrations of each permafrost-based library after library preparation;
obtained using a Qubit Florometer.

21

Table 7. DNA concentrations of each active layer-based library after library preparation;
obtained using a Qubit Florometer.

CONCLUSIONS AND FUTURE WORK
DNA and RNA extractions were successfully prepared from the microbial communities
present in the soil compositions prepared. RNA was not extracted at high enough concentrations
for further analyses, although DNA was extracted in high enough concentrations for further
processing. The DNA extracts underwent a series of processing steps, including attachment of
adaptors for Illumina sequencing, attachment of known nucleotide barcodes, and qPCR analysis.
Analysis of qPCR showed minimal results in the permafrost-based samples due to low
concentration of DNA present. This result is most likely due to low inputs of DNA in the earlier
PCR reactions during library preparation and improper ligation of adaptors due to this low DNA
input. Improved methodology of the PCR preparation by re-running libraries with varied inputs
of reaction reagents and DNA may improve quality of libraries prepared.

22

Metagenomic sequencing of the libraries still must be conducted. Libraries prepared are now
available to be sequenced. Sequencing and metagenomic software analysis of the libraries will
provide insight into hypothesis raised about functional gene composition of microbial
communities present in permafrost soil. Soil samples that have incubated for 12 weeks are
expected to have proliferated in unique ways as compared to samples that did not undergo thaw.
Metagenomic sequencing will identify functional gene abundance differences between the soil
samples that have thawed for 12 weeks compared to samples with no thaw. These differences
in functional gene abundance will give insight into the microbial species present in the
respective active and permafrost layers, as well as aim to identify how microbial communities are
responding to this change in environment.

23

LITERATURE CITED
Brown et al. (1997). International Permafrost Association. Carleton University Department of
Geography and Environmental Studies. https://www.permafrost.org/data/.
Carpino O, Haynes K, Connon R, Craig J, Devoie É, Quinton W. (2021). Long-term climateinfluenced land cover change in discontinuous permafrost peatland complexes.
Hydrology and Earth System Sciences. doi:https://doi.org/10.5194/hess-25-3301
https://hess.copernicus.org/articles/25/3301/2021/.
De Maayer, P., Anderson, D., Cary, C., & Cowan, D. A. (2014). Some like it cold:
understanding the survival strategies of psychrophiles. EMBO Reports, 15, 508–517.
https://doi.org/10.1002/embr.201338170
Green, E. (2024). Contig. Genome.gov. https://www.genome.gov/genetics- glossary/Contitg
Hofer, U. (2018). The majority is uncultured. Nature Reviews Microbiology, 16, 716–717.
https://doi.org/10.1038/s41579-018-0097-x
Jansson K, Taş N. (2014). The microbial ecology of permafrost. Nature Reviews Microbiology.
12, 414–425. doi:https://doi.org/10.1038/nrmicro3262.
Kuiper, M. J., Morton, C. J., Abraham, S. E., & Gray-Weale, A. (2015). The biological
function of an insect antifreeze protein simulated by molecular dynamics. Elife.
https://doi.org/10.7554/elife.05142
Lluis M. Martínez. (2015). Magnetic DNA Purification: History and recent developments.
Sepmageu. https://www.sepmag.eu/blog/magnetic-dna-purification-history-recentdevelopments.
Mackelprang R, Saleska SR, Jacobsen CS, Jansson JK, Taş N. (2016). Permafrost Meta-Omics
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and Climate Change. Annual Review of Earth and Planetary Sciences. 44, 439–
462.doi:https://doi.org/10.1146/annurev-earth-060614-105126.
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