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Next-Generation Sequencing: Current Technologies and Applications

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DOI: 10.1007/978-3-030-06088-6_39

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 Next-Generation Sequencing:
Current Technologies 39
and Applications

Dwarakanath Srinivas and Harsh Deora

Abbreviations HMP Working Group et al. 2009; Huttenhower

et al. 2012). Although fungi contribute less than
ALS Amyotrophic lateral sclerosis 0.1% of the total microbiome, they contribute a
CNS Central nervous system major role in the various physiological and path-
HMP Human Microbiome Project ological processes of the body.
ITS Internal transcribed spacer More importantly, etiological diagnosis of
MetaHIT Metagenomics of the Human Intestinal inflammatory disorders of the central nervous
Tract system (CNS) is a major challenge, with more
NGS Next-generation sequencing than 50% cases going undiagnosed (Glaser et al.
SNP Single-nucleotide polymorphism 2006). Next-generation sequencing (NGS) and
metagenomics provide information regarding not
only the transcriptome of the human tissue but
39.1 Introduction also of the microbiome that resides in it (Salzberg
et al. 2016). 16s rRNA gene-based pathogen
The human body, apart from human cells, is com- identification is exclusive to prokaryotes, and
posed of microbial flora, which plays an impor- hence deep sequencing of the total DNA or RNA
tant role in various physiological processes, has been utilized to detect even the rarest patho-
called as the human microbiome (Zoll et al. gens present in the microbiome (Wylie et al.
2016). Population-scale projects such as the 2013).
Human Microbiome Project (HMP) and the
Metagenomics of the Human Intestinal Tract
(MetaHIT) project have provided a glimpse into 39.2 Current Technologies
the microbial composition of different mucosa,
like the skin, the gastrointestinal tract, the respi- NGS involves sequencing of the entire microbi-
ratory tract, and the urogenital tract (The NIH ome of the sample. The application of NGS is
still in infancy when it comes to clinical mycol-
D. Srinivas (*) ogy. However, giant strides are being made in
Department of Neurosurgery, National Institute of mycology research that may soon translate into
Mental Health and Neurosciences (NIMHANS), its application to routine diagnostic mycology.
Bangalore, India
H. Deora
National Institute of Mental Health and
Neurosciences (NIMHANS), Bangalore, India

© Springer Nature Switzerland AG 2019 471

M. Turgut et al. (eds.), Fungal Infections of the Central Nervous System,
https://doi.org/10.1007/978-3-030-06088-6_39
472
39.3 Clinical Mycology
In addition to data obtained from population-scale
projects such as HNP and MetaHIT, Bittinger
et al. has demonstrated that the relative proportion
of bacteria and mycobiota in the lung can be used
to differentiate infection from colonization
(Bittinger et al. 2014). However, fungal species
constitute only 0.1–1% of the human microbiome
and even less so in the CNS. This would imply that
a level of 1012–1014 nucleotides per sequencing run
per sample is required to even detect subspecies of
the fungi. This means that current available
sequencers such as Illumina Miseq, Ion Torrent
PGM, and HiSeq are far from the desired sensitiv-
ity required to diagnose fungal infections.
Using mycobiomes obtained by sequencing
only amplicons of internal transcribed spacer
(ITS) of the fungal ribosomal genes, using
­platforms like Illumina NextSeq, it may be possi-
ble to extract information in clinical samples. This
would provide a relative composition of the micro-
biological flora and thus provide an opportunity
for application to clinical mycology. Especially in
the CNS, the relative proportion of fungi would
also give an indication of the severity of the fungal
infection. Salzberg et al. in a prospective pilot
study applied NGS in combination with computa-
tional analysis to detect the presence of pathogenic
microbes in brain and spinal cord biopsies from
ten patients with findings suggestive of infection
but clinical and microbiological studies yielding
inconclusive results (Salzberg et al. 2016). They
were able to detect infectious processes in eight of
the ten cases, thus providing evidence that NGS
can dramatically improve our ability to detect or
rule out a wide range of CNS pathogens. This is
especially applicable to cases where conventional
microbiology has been negative or takes too much
time to be clinically viable (Salzberg et al. 2016).
39.4 Research Mycology
NGS has been used frequently for research pur-
poses. Options include single-nucleotide poly-
morphisms (SNPs), microsatellite analysis,
amplicon sequencing of ribosomal ITS, and
whole genome sequencing. While microsatellite
D. Srinivas and H. Deora
analysis and SNPs are commonly used, ribo-
somal ITS and whole genome sequencing are the
most sensitive in distinguishing various fungal
pathogens (Araujo 2014; Dannemiller et al.
2014). Decker et al. showed that a NGS-based
diagnostic approach was the best in patients with
septic shock. They also demonstrated that inva-
siveness mycoses could be distinguished from
colonization, thus indicating the need for antifun-
gal therapy (Decker et al. 2017). Alonso et al.
suggested that amyotrophic lateral sclerosis
(ALS) may be a fungal disease and demonstrated
a variety of fungal species in each case of ALS
with the help of NGS. They even suggest that
severity and evolution of the disease may vary
from patient to patient in accordance with the
fungal species (Alonso et al. 2017).
Azole resistance has been demonstrated in
fungal infections including itraconazole, voricon-
azole, posaconazole, and isavuconazole, espe-
cially after long antifungal azole treatment (Bueid
et al. 2010; Lockhart et al. 2011; Howard et al.
2009; Snelders et al. 2008; Verweij et al. 2009a).
Mechanisms include efflux pumps, which reduce
intracellular drug concentration, increased azole
target enzyme production, and adaptation of tar-
get site of demethylases active in sterol synthetic
pathways. Forward and reverse genetic approaches
have demonstrated genes involved in Cryptococcus
neoformans life cycle. Ianiri and Idnurm evalu-
ated 35 genes required for viability in ascomyce-
tes and drug resistance, demonstrating genes
involved in ergosterol biosynthetic pathway
(Ianiri and Idnurm 2015). Similarly, Candida iso-
lates resistant to azoles have shown mutations in
genes involved in formation of demethylases and
efflux pumps (Garnaud et al. 2015).
Aspergillus fumigatus is an opportunistic fun-
gus causing a variety of diseases in immuno-­
compromised hosts, including invasive
aspergillosis. Azole resistance is widespread in
this species with resistant species being isolated
form the environment (Snelders et al. 2009;
Verweij et al. 2009b). Insight into the methods of
azole resistance in this group has been investi-
gated with the help of whole genome sequencing
on isolated strains of Aspergillus from patients
with long-term azole resistance. Elucidated mech-
anism points to mutations in Cyp51 A protein.
39 Next-Generation Sequencing: Current Technologies and Applications 473

Cyp51 A protein is a demethylase involved in stress and is regulated with epigenetic mecha-
ergosterol synthesis which is a component of the nisms like DNA hydroxylation-methylation.
fungal cellular wall and the main substrate for Cytosine methylation or hydroxylation can be
demethylase inhibitors like azoles (Latgé 1999). studied by the chemical conversion using bisul-
Mutations like amino acid substitution including fate of cytosine into uracil.
G54A, P216L, M220V, Y121F, and duplication Computational analysis along with NGS is
of 34 and 46 bp nucleotides in the promoter powerful tool to study/diagnose fungal infec-
region of CYP51A gene have been found to be tions. Transcriptome analysis studies gene
responsible for azole resistance. While former expression by growing fungal cells after messen-
mutations are just a marker for azole resistance, ger RNA are extracted from cells and sequenced
duplication in the promoter region leads to after conversion to complementary DNA. Thus,
increase in Cyp51 A protein synthesis. the relative abundance of messenger RNA grown
Other more obscure mechanisms have been after and before exposure to azoles provides evi-
reported. Camps et al. reported four Aspergillus dence to the change in gene expression because
fumigatus isolates in which two species devel- of these compounds.
oped azole resistance after prolonged therapy and Thus, while whole genome sequencing over-
could not be explained by Cyp51 A protein muta- comes much of the limitations of currently
tions (Camps et al. 2012). They followed these available sequencers like Illumina NextSeq and
changes with Whole genome sequencing of the HiSeq and provides insight into mechanisms of
isogenic Aspergillus fumigatus species and azole resistance, transcriptome analysis and
revealed several non-synonymous mutations. To DNA methylation provide proof of gene expres-
correlate mutations with phenotypes, sexual sion changes with azole exposure. These are the
crossing experiments were done on the progeny tools currently available for research in
of azole resistance phenotype. These revealed mycology.
that azole resistance was associated with a P88L
amino acid substitution in the CCAAT-­binding
transcription factor complex subunit HapE. Also, 39.5 Conclusion
the HapE P88L mutation caused an increased
CYP51A gene expression thus causing Accuracy, speed, and sensitivity are the USP of
resistance. NGS. It is especially valuable in fungal infec-
Similar study done by Fraczek et al. in tions as these are notoriously difficult to grow in
Aspergillus fumigatus species lacking Cyp51 A culture and thus offer “culture-independent”
mutation revealed 20 potential azole transporter mechanisms of diagnosis. In addition, these offer
genes (Fraczek et al. 2013). In one, CYP51A similar if not better information considering the
expression was increased 500 times in the pres- application of NGS to detect azole resistance,
ence of azoles. Others demonstrated an increase distinguishing infection and colonization and
in CDR1B efflux transporter gene and in one out guiding treatment. Thus, “next” generation needs
of five isolates, a P216L amino acid substitution to be incorporated to current diagnostic
was found in Cyp51A in conjunction with several methodologies.
other non- synonymous mutations and deletions
of clusters of genes (Hagiwara et al. 2014). These
authors thus concluded that the cdr1B efflux
References
transporter is associated with Cyp51A-­
independent azole resistance. Alonso R, Pisa D, Fernandez-fernandez AM, Rabano
While whole genome sequencing has offered A, Carrasco L. Fungal infection in neural tissue of
us insight into the mechanisms of azole resis- patients with amyotrophic lateral sclerosis. Neurobiol
tance, other NGS tools have demonstrated Dis. 2017;108:249–60.
Araujo R. Towards the genotyping of fungi: methods,
changes in genetic expression due to environ- benefits and challenges. Curr Fungal Infect Rep.
mental changes. Gene expression changes with 2014;8:203–10.
474
Bittinger K, Charlson ES, Loy E, Shirley DJ, Haas AR,
Laughlin A, et al. Improved characterization of medi-
cally relevant fungi in the human respiratory tract
using next-generation sequencing. Genome Biol.
2014;15:487.
Bueid A, Howard SJ, Moore CB, Richardson MD,
Harrison E, et al. Azole antifungal resistance in
Aspergillus fumigatus: 2008 and 2009. J Antimicrob
Chemother. 2010;65:2116–8.
Camps SMT, Dutilh BE, Arendrup MC, Rijs AJMM,
Snelders E, Huynen MA, et al. Discovery of a HapE
mutation that causes azole resistance in Aspergillus
fumigatus through whole genome sequencing and
sexual crossing. PLoS One. 2012;7:e50034.
Dannemiller KC, Reeves D, Bibby K, Yamamoto N, Peccia
J. Fungal high-throughput taxonomic i­dentification
tool for use with next-generation sequencing
(FHiTINGS). J Basic Microbiol. 2014;54:315–21.
Decker SO, Sigl A, Grumaz C, et al. Immune-response
patterns and next generation sequencing diagnostics
for the detection of mycoses in patients with septic
shock—results of a combined clinical and experi-
mental investigation. Int J Mol Sci. 2017;18(8):1796.
https://doi.org/10.3390/ijms18081796.
Fraczek MG, Bromley M, Buied A, Moore CB, Rajendran
R, Rautemaa R, et al. The cdr1B efflux transporter is
associated with non-cyp51a-mediated itraconazole
resistance in Aspergillus fumigatus. J Antimicrob
Chemother. 2013;68:1486–96.
Garnaud C, Botterel F, Sertour N, Bougnoux M, Dannaoui
E, Larrat S, et al. Next-generation sequencing offers
new insights into the resistance of Candida spp. to
echinocandins and azoles. J Antimicrob Chemother.
2015;70:2556–65.
Glaser CA, Honarmand S, Anderson LJ, et al. Beyond
viruses: clinical profiles and etiologies associ-
ated with encephalitis. Clin Infect Dis. 2006;43:
1565–77.
Hagiwara D, Takahashi H, Watanabe A, Takahashi-­
Nakaguchi A, Kawamoto S, Kamei K, et al. Whole-­
genome comparison of Aspergillus fumigatus strains
serially isolated from patients with Aspergillosis. J
Clin Microbiol. 2014;52:4202–9.
Howard SJ, Cerar D, Anderson MJ, Albarrag A, Fisher
MC, et al. Frequency and evolution of azole resistance
in Aspergillus fumigatus associated with treatment
failure. Emerg Infect Dis. 2009;15:1068–76.
View publication stats
D. Srinivas and H. Deora
Huttenhower C, Gevers D, Knight R, Abubucker S,
Badger JH, Chinwalla AT, et al. Structure, func-
tion and diversity of the healthy human microbiome.
Nature. 2012;486:207–14.
Ianiri G, Idnurm A. Essential gene discovery in the basidio-
mycete Cryptococcus neoformans for antifungal drug
target prioritization. MBio. 2015;6(2):e02334–14.
Latgé JP. Aspergillus fumigatus and aspergillosis. Clin
Microbiol Rev. 1999;12:310–50.
Lockhart SR, Frade JP, Etienne KA, Pfaller MA, Diekema
DJ, et al. Azole resistance in Aspergillus fumigatus iso-
lates from the ARTEMIS global surveillance is primar-
ily due to the TR/L98H mutation in the cyp51A gene.
Antimicrob Agents Chemother. 2011;55:4465–8.
Salzberg SL, Breitwieser FP, Kumar A, Hao H, Burger
P, Rodriguez FJ, et al. Next-generation sequenc-
ing in neuropathologic diagnosis of infections
of the nervous system. Neurol Neuroimmunol
Neuroinflamm. 2016;3:e251. https://doi.org/10.1212/
NXI.0000000000000251.
Snelders E, van der Lee HA, Kuijpers J, Rijs AJ, Varga
J, et al. Emergence of azole resistance in Aspergillus
fumigatus and spread of a single resistance mecha-
nism. PLoS Med. 2008;5:e219.
Snelders E, Huis In ’t Veld RA, Rijs AJ, Kema GH,
Melchers WJ, Verweij PE. Possible environmen-
tal origin of resistance of Aspergillus fumiga-
tus to medical triazoles. Appl Environ Microbiol.
2009;75(12):4053–7.
The NIH HMP Working Group, Peterson J, Garges S,
Giovanni M, McInnes P, Wang L, et al. The NIH human
microbiome project. Genome Res. 2009;19:2317–23.
Verweij PE, Howard SJ, Melchers WJ, Denning
DW. Azole- resistance in Aspergillus: proposed
nomenclature and breakpoints. Drug Resist Updat.
2009a;12:141–7.
Verweij PE, Snelders E, Kema GH, Mellado E, Melchers
WJ. Azole resistance in Aspergillus fumigatus: a side-­
effect of environmental fungicide use? Lancet Infect
Dis. 2009b;9:789–95.
Wylie KM, Weinstock GM, Storch GA. Virome genom-
ics: a tool for defining the human virome. Curr Opin
Microbiol. 2013;16:479–84.
Zoll J, Snelders E, Verweij PE, Melchers WJG. Next-­
generation sequencing in the mycology lab. Curr
Fungal Infect Rep. 2016;10:37–42. https://doi.
org/10.1007/s12281-016-0253-6.