Respiratory Treatment and Prevention (Advances in Experimental Medicine and Biology)

et al. 2014 ) and the identification of cyclovirus
CyCV-VN as a novel etiological factor in
encephalitis (Tan le et al. 2013 ).
In the present investigation, sensitivity of
NGS, preceded by isothermal RNA amplification
(Ribo-SPIA), was 10^3 copies for HSV-1 and 10^2
copies for HIV in CSF. A similar sensitivity
(using Ribo-SPIA and Illumina platform HiSeq)
with respect to RNA viruses has been reported by
Malboeuf et al. ( 2013 ), who detected 10^2 copies
of HIV, West Nile virus (WNV), and the respira-
tory syncytial virus (RSV) in plasma samples. A
head-to-head comparison of sensitivity of differ-
ent NGS protocols is rarely done, but the
Illumina platforms show the highest efficiency
and sequencing depth when compared to such
other NGS platform as Ion PGM Torrent and
pyrosequencing-based platforms (Frey

et al. 2014 ; Lecuit and Eloit 2014 ; Cheval

et al. 2011 ).

Nonetheless, short reads generated by

Illumina platforms make the assembly of a full

length of microbial genomes challenging

(Quinones-Mateu et al. 2014 ; Virgin and Todd

2011 ). That is particularly important for the

detection of unknown pathogens, wherede novo

assembly is necessary (Miyamoto et al. 2014 ). In

addition, high coverage rate of a reconstructed

genome may facilitate the interpretation of

results. Currently, there are no established

criteria for the detection of pathogens by NGS.

Palacios et al. ( 2008 ) have identified astrovirus

infection in a transplant patient after detection of

14 sequences that constituted only 0.0135 % of

all reads, while in a study of Feng et al. ( 2008 )

the identification of Merkel cell polyomavirus

Table 3 Most frequently identified species/genera in the cerebrospinal fluid (CSF) sample from a control patient with
motor neuron disease (MND) and in two water samples (W1 and W2)

Sample Viruses Bacteriaa Fungia Protozoaa

MND None Corynebacterium

(96,345)

Funneliformis (19,789) Besnoitia (54,232)

Pseudomonas (82,301) Malassezia (14,005) Nannochloropsis (2028)

Staphylococcus

(64,799)

Glomus (13,976) Phytophthora (1647)

Bacillus (61,580) Cladosporium (10,127) Plasmodium (1270)

Escherichia (41,394) Melampsora (5663) Albugo (1197)

W1 Influenza C (670) Psychrobacter

(307,551)

Phialocephala subalpine

(1202)

None

HCV (36) Staphylococcus

(128,538)

Malassezia sympodialis

(731)

Pepino Mosaic

Virus (3)

Corynebacterium

(75,297)

Leptosphaeria maculans

(682)

Micrococcus (35,496) Parastagonospora nodorum

(680)

No taxonomic data

(29,436)

Arthroderma obtusum (562)

W2 None Acinetobacter

(215,184)

Malassezia restricta (5967) Albugo laibachii (4645)

Streptococcus

(103,489)

Malassezia globose (5882) Phytophthora megasperma

(2835)

Corynebacterium

(95,912)

Penicillium rubens (5828) Nannochloropsis oceanica

(2786)

Micrococcus (90,144) Polycephalomyces formosus

(4732)

Peronospora aquatic (2427)

Staphylococcus

(75,621)

Aspergillus fumigatus

(4505)

Amphifilidae sp. H-1
(1736)
HSV-1 herpes simplex virus type 1,HIVhuman immunodeficiency virus,HCVhepatitis C virus
aFive most numerous genera

Sensitivity of Next-Generation Sequencing Metagenomic Analysis for Detection... 59