A comparative study of small RNAs in Toxoplasma gondii of distinct genotypes
Jielin Wang
0
Xiaolei Liu
0
Boyin Jia
0
Huijun Lu
0
Shuai Peng
0
Xianyu Piao
1
Nan Hou
1
Pengfei Cai
1
Jigang Yin
0
Ning Jiang
0
Qijun Chen
0
1
0
Key Laboratory of Zoonosis, Ministry of Education, Jilin University
,
Xi An Da Lu 5333, Changchun 130062
,
China
1
MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College
,
Beijing
,
China
Background: Toxoplasma gondii is an intracellular parasite with a significant impact on human health. Inside the mammalian and avian hosts, the parasite can undergo rapid development or remain inactive in the cysts. The mechanism that regulates parasite proliferation has not been fully understood. Small noncoding RNAs (sncRNA) such as microRNAs (miRNAs) are endogenous regulatory factors that can modulate cell differentiation and development. It is anticipated that hundreds of miRNAs regulate the expression of thousands of genes in a single organism. SncRNAs have been identified in T. gondii, however the profiles of sncRNAs expression and their potential regulatory function in parasites of distinct genotypes has largely been unknown. Methods: The transcription profiles of miRNAs in the two genetically distinct strains, RH and ME49, of T. gondii were investigated and compared by a high-through-put RNA sequencing technique and systematic bioinformatics analysis. The expression of some of the miRNAs was confirmed by Northern blot analysis. Results: 1,083,320 unique sequences were obtained. Of which, 17 conserved miRNAs related to 2 metazoan miRNA families and 339 novel miRNAs were identified. A total of 175 miRNAs showed strain-specific expression, of which 155 miRNAs were up-regulated in RH strain and 20 miRNAs were up-regulated in ME49 strain. Strain-specific expression of miRNAs in T. gondii could be due to activation of specific genes at different genomic loci or due to arm-switching of the same pre-miRNA duplex. Conclusions: Evidence for the differential expression of miRNAs in the two genetically distinct strains of T. gondii has been identified and defined. MiRNAs of T. gondii are more species-specific as compared to other organisms, which can be developed as diagnostic biomarkers for toxoplasmosis. The data also provide a framework for future studies on RNAi-dependent regulatory mechanisms in the zoonotic parasite.
-
Background
Toxoplasma gondii is an obligatory intracellular
parasite that infects a wide range of hosts,including humans,
animals and birds. It is considered to be one of the
most widely distributed protozoan parasite with a
seroprevalence in humans of up to 30% worldwide [1]. T. gondii
is the etiological agent of toxoplasmosis which can be
either life-threatening or long-term chronic infection.
The life cycle of Toxoplasma gondii is unusual in that
the parasite is capable of indefinite proliferation in the
hosts with either a sexual or an asexual cycle. The sexual
cycle occurs only in the hosts of a feline species. The
asexual cycle can occur in virtually any warm-blooded
animals, which act as the intermediate hosts, ranging
from chicken to baleen whales and humans. In the
parasites life cycle, there are three fundamental
morphotypes, named tachyzoites, bradyzoites and sporozoites.
The development of T. gondii in the intermediate host
involves an initial phase with a rapid proliferation of the
tachyzoites, followed by the formation of tissue cysts
containing slowly dividing or resting bradyzoites.
While the global population structure of T. gondii
awaits further elucidation [2], three clonal lineages
(Type I, II, and III) of T. gondii which comprise the
majority of strains in both North America and Europe
[3-5]. Recently, a fourth clonal lineage, designated
haplogroup 12, has been identified based on isolates that are
common in wild animals in the United States [6]. The
virulence of T. gondii is normally defined based on the
LD50 in mice. Type I strain has been regarded as a
more virulent strain in mice with an LD50 <10 [4,7].
In contrast, type II (e.g., ME49) and type III strains
are less lethal in mice (LD50 > 100) and the infections
are usually less severe or asymptomatic [8]. The clonal
lineages also differ in a number of phenotypes such as
growth rate, efficiency in migration and transmigration
in tissues [7,9].
Several studies have been carried out with aims to
determine differential gene expression at various
lifecycle stages of T. gondii [10-13]. Likewise, efforts were
taken to reveal the factors that may modulate the
virulence of T. gondii. ROP5, ROP16 and ROP18, which are
rhoptry-derived factors, and their expression was found
to correlate with parasite virulence [14-18].
Identification of strain-specific regulatory elements responsible
for the distinct genotype will not only facilitate our
understanding of parasite biology, but may also elucidate
the association between genetically defined
subpopulations (strains) and disease severity.
In recent years, the discovery of numerous small RNAs
has increased our knowledge in post-transcriptional gene
regulation in cell development and other biological
processes. Small RNAs, such as microRNA (miRNA), small
interfering RNA (siRNA), and Piwi-associated RNA
(piRNA), are regulatory elements that can modulate gene
expression at the post-transcriptional level. MiRNAs,
a class of 22 nucleotide small-RNA sequences that
participate in the post-transcriptional regulation of gene
expression [19], have been known to play critical roles in
diverse biological processes, including development, viral
defense, metabolism, and apoptosis [20-23]. The primary
transcripts (pri-miRNAs) of miRNA genes are generated
by either RNA polymerase II [24] or RNA polymerase III
[25]. A single pri-miRNA may contain from one to six
miRNA precursors. They are processed by RNase
complexes (Drosha and DGCR8) into 70 nucleotide
fragments with a stem-loop structure called pre-miRNA.
The pre-miRNAs are then exported to the cytoplasm by
Exportin-5 [26] and the hairpin is cleaved by the RNase
III enzyme (Dicer) to miRNA duplexes [27]. A single
miRNA can silence a number of genes while one gene
can be targeted by several miRNAs [28,29]. Previous
studies have found that T. gondii has a complete
machinery for small RNA generation and small RNA-mediated
gene regulation [30]. There are mainly two classes of
small regulatory RNAs derived from the T. gondii
genome, namely miRNA and rdsRNA. And it was found that
rdsRNAs were consistently more abundant in the highly
virulent Toxoplasma isotype-I (RH) than in other two
strains (RPU and CTG). Further, it was recently reported
that the Argonaute of T. gondii (TgAgo) is methylated
and its activity is Mg2+-dependent [31]. However the
differential expression of miRNA in parasites of distinct
genotypes has not been well investigated.
In this study, we systematically studied the expression of
miRNAs in type I (RH) and type II strain (ME49) T. gondii
by high-through-put RNA sequencing technology and
bioinformatic analysis. A number of T. gondii specific
miRNAs was identified. Meanwhile, differentially expressed
miRNAs between the two strains of T. gondii were
detected. The results demonstrated profound differences
between the two strains of T. gondii in miRNA expression.
Methods
Parasites
Tachyzoites of T. gondii RH strain and ME49 strain were
propagated in mice. The tachyzoites were purified by
density-gradient centrifugation on Percoll [32]. The
study of using laboratory animals was reviewed and
approved by the Ethical Committee of Jilin University.
RNA isolation
Total RNA of T. gondii (both RH and ME49 strain) was
prepared using Trizol reagent (Invitrogen, SF, USA)
according to the manufacturers protocol. The integrity
of total RNA was examined by standard agarose gel
electrophoresis, and RNA purity was reflected by the 260/
280 nm absorbance ratio and the concentration was
determined using a Nanodrop 1000 machine (Thermo
Scientific CA, USA). The purified total RNA was stored
at-80C until use.
Construction of small RNA libraries and sequencing
For small RNA library construction and deep
sequencing, RNA samples from RH and ME49 strains of
T. gondii were prepared as follows: for each strain, equal
quantities (10 g) of RNA isolated from tachyzoites were
pooled. Small RNA molecules in the range of 1830 nt
RNA was purified after polyacrylamide gel
electrophoresis (PAGE) and ligated with proprietary adapters to
the 50 and 30 termini. The samples were used as
templates for cDNA synthesis. The cDNA was amplified
to produce sequencing libraries which were subjected
to Solexas sequencing-by-synthesis method. Sequencing
was carried out at the Beijing Genomics Institute (BGI,
China). Two separated runs with two batches of RNAs
were carried out in the sequencing. Only sequences with
high quality were included in the analysis.
Mapping sequence reads to the reference genome
Individual sequence reads with the base quality scores
were produced by Solexa. Clean reads were obtained
after removing of the low quality reads, such as adaptor
null reads, insert null reads, 50 adaptor contaminants,
and reads with polyA tail and ambiguous bases. Adapter
sequences were trimmed from both ends of clean reads
before analysis. All identical sequences were counted and
eliminated from the initial data set. The resulting set of
unique sequences with associated read counts was referred
to as sequence tags. The unique reads were mapped onto
the T. gondii genome (http://www.toxodb.org) using the
program SOAP (http://soap.genomics.org.cn) [33].
Bioinformatic analysis of T. gondii small RNAs
Sequences were, at the first step, searched against
Metazoa mature miRNA of Sanger miRBase allowing two
mismatches to identify homologs of known Metazoa
miRNAs using the program Patscan [34]. Sequence tags
of more than 5 reads that matched perfectly or
nearperfectly (no more than 2 mismatches and mismatch
not positioned in seed region) to metazoan mature
miRNAs were assumed to be conserved miRNA candidates.
Secondly, the remaining sequences were screened
against the non-coding RNA database [35] and the T.
gondii genome database, and some non-coding RNAs
such as rRNA, tRNA and snoRNAs were identified and
filtered out for further analysis. Thirdly, We searched for
the inverted repeats (step loops or hairpin structure)
among the remained sequences which did not match to
the miRNA database by using the software Einverted of
Emboss [36] with the parameters following parameters:
threshold = 30, match score = 3, mismatch score = 3, gap
penalty = 6, and maximum repeat length = 240 as
described [37]. Each inverted repeat was extended 10 nt on
each side, and the secondary structure of the inverted
repeat was predicted by RNAfold [38]. Unique reads in the
inverted repeats were evaluated by MirCheck [37] using
modified parameters that are more suitable for organisms
such as worms and protozoa. Finally, miRNA precursors
that passed MirCheck were inspected manually in order to
remove the false prediction (Additional file 1: Figure S1).
Further, miRNAs with the same sequences derived from
pre-miRNAs (the sequences may not be completely
the same) located at different genomic loci are
indicated with an additional dash-number suffix. Sequences
of T. gondii-specific miRNAs have been deposited in the
miRNA database (miRBase).
MiRNAs with statistically significant differences in relative
abundance (as reflected by TPM values) between the two
libraries (corresponding to the two distinct strains of the
parasites) were analyzed with IDEG6 and edger [39]. We
used the general Chi-square method as it is most frequently
used in such analysis [40,41]. MiRNA with a P value
0.01 were deemed to be significantly different between
the samples of the two distinct strains of parasites.
Northern blot analysis of miRNA expression
Twenty micrograms of total RNA was loaded in each
well of a 12.5% polyacrylamide gel containing 42%
urea and run in 0.5 TBE buffer. Following the
electrophoresis, RNA was transferred by capillary
transferring to a Hybond-N + nylon membranes (GE
Healthsystems, Uppsala, Sweden). After UV cross-linking, the
membranes were baked for 1 h at 80C. Probes
complementary to small RNA sequences were end-labeled
with DIG at 50 Termini (TaKaRa, Dalian, China).
Prehybridization of the membrane was performed by
overnight incubation at 53C followed by hybridization
overnight at the same temperature in Northernmax
Hybridization buffer (Ambion, CA, USA). After
hybridization, the membranes were washed four times for
30 min in 2SSC, 0.05% SDS and twice for 15 min in
0.1SSC, 0.1% SDS at RT. Chemiluminescent signal
was detected using a DIG Detection Kit (Roche,
Germany) following the manufacturers instructions. The
oligonucleotide probes used for hybridization are as
follows:
Results
An overview of the small RNA sequencing results
A total of 8,738,870 and 10,759,107 sequence reads were
obtained from the ME49 and RH libraries, respectively.
There is no major difference in total numbers of low
and high quality reads between the two libraries
(Additional file 2: Table S1). After removal of the low
quality reads, 7,149,051 (ME49) and 8,494,754 (RH) clean
reads were obtained, which contained 247,346 (ME49) and
867,853 (RH) library-specific (unique) reads, respectively
(Figure 1).
The clean unique reads described above were mapped
to the draft genome of T. gondii (http://toxodb.org/toxo/
showApplication.do) [33]. 6,068,932 and 3,088,774
nonredundant total reads from ME49 and RH were perfectly
mapped onto the T. gondii genome, respectively
(Additional file 3: Table S2). Among these reads, 51,832
(76.65% of total reads in ME49 library) displayed
strainspecificity in ME49, while in RH the number of
strainspecific reads was 117,315 (88.14% of total reads in RH
library), the number of small RNA reads identified in the
two strains was 15,791, which accounted for 11.86% and
Figure 1 Work flow for profiling of small RNAs after high-through-put sequencing. Approaches and numbers of sequence tags obtained
after each step were shown for both strains. The simple pie chart shows the number of strain-specific and common miRNAs identified in the two
libraries. More miRNAs (155) were identified in RH strain than that in ME49. The number of miRNAs that commonly expressed in the two strains
is 181.
23.35% of the total reads of RH and ME49, respectively
(Figure 2). The length of small RNAs varied from 18 to
30 nt in the two strains. However, the length
distributions of small RNAs were significantly different (Figure 3,
Figure 2 Comparison of small RNAs sequences identified in the
two strains of T. gondii at unique level. Of the small RNAs
identified in the two strains of T. gondii, strain-specific small RNAs
were much more (88.14% of RH strain and 76.65% of ME49 strain)
than that shared by the two strains (11.86% of RH strain and 23.35%
of ME49 strain).
Additional 4: Figure S2 and Additional file 5: Table S3).
A majority of small RNAs in ME49 was 21 nt in length,
whereas small RNAs of 26 nt were the most abundant in
RH strain. The small RNAs were further categorized
into, based on the sequence characteristics, rRNAs,
tRNAs, small nuclear RNAs (snRNAs) and other ncRNA
after BLASTN searches against the Sanger Rfam
database release 9.0 (Additional file 3: Table S2). The
proportions of small ncRNAs in the two libraries (ME49 and
RH) were shown in Figure 2 and Figure 4A, B.
Identification of miRNAs in T. gondii
The proportions of miRNAs in the two libraries
accounted between 5-8% of the total reads of the small
ncRNAs (Figure 4 A, B). In total, 17 conserved miRNAs
were found based on the consensus seed region (28 nt
in 50 end of mature miRNA) identical to that of Homo
sapiens, Mus musculus and Pongo pygmaeus (Figure 5),
which were recognized by mRNAs through base pairing
comparison [42]. In addition to the conserved miRNAs,
we also found 339 species-specific miRNAs (novel
miRNAs) of T. gondii. Moreover, 20 (5.6%) of 356
miRNAs were from the ME49 library, while 155 (43.6%)
were from RH library, and 181 (50.8%) of the miRNAs
were found in both libraries (Figure 1, Additional file 6:
Table S4 and Additional 7: Table S5). The number of
Figure 3 The length distribution of small ncRNAs in the library of ME49 (blue) and RH (red) of T. gondii. The dominant sncRNA expressed
in ME49 strain is 21 nt in length, while the 26 nt sncRNAs were dominant in RH strain.
Characterization of the expression of T. gondii miRNAs
Based on their genomic locations, T. gondii miRNAs
were categorized into three types (Additional file 6:
Table S4 and Additional file 7: Table S5) named intronic,
intergenic and UTR-derived miRNAs. The numbers of
miRNAs derived from the 3 genomic regions were 33,
305 and 18 respectively. No miRNA genes were found to
be located in exons. Thus miRNA genes were
predominantly intergenic in T. gondii. This observation was in
agreement with a previous study on Schistosoma
japonicum which suggested that most miRNA genes have
their own control elements (or promoters) in the genome
[43]. Further, it was found that a miRNA can be
generated from several pre-miRNAs encoded by genes located
in different genomic loci. Thus miRNAs with the same
sequence but derived from different pre-miRNAs were
named with an additional dash-number suffix (Additional
file 6: Table S4 and Additional file 7: Table S5).
Comparison of the novel miRNAs expressed in ME49
and RH revealed strain-specific expression patterns in
the two distinct strains of T. gondii. For instance, the
number of reads of tgo-novel-40 in ME49 was 4000
times more than that in RH. Similarly, the expression
level of tgo-novel-1-1-5p, tgo-novel-12-1, tgo-novel-41
and tgo-novel-15-1 was significantly higher in ME49
strain. On the contrary, tgo-novel-14-2, was highly
expressed in RH, the reads number was about 10 times
Figure 4 Percentages of small ncRNAs in the two libraries
(A. ME 49 strain, B. RH strain) of T. gondii. A majority of the
sncRNAs in the two libraries is unknown. The proportion of miRNAs
in ME49 and RH strain is 8.08% and 5.30% respectively.
Figure 5 Alignment of tgo-miR-574 sequence with homologues
from other organisms. The seed sequences are shadowed in dark
colour. Mmu-miR-574-5p, mmu-miR-1187 and mmu-Mir-466i-5p are
miRNAs identified in mice. Has-miR-574-5p is a miRNA identified in
humans.
as much as that in ME49 (Additional file 7: Table 5 and
Additional file 8: Table S6).
Previous studies have found that mature miRNAs can
be derived from both arms of a pre-miRNA hairpin
[44,45]. In this study, we found 178 miRNAs were
derived from just one hairpin arm, of which 81 mature
miRNAs were located at 30 arm and 97 were located at
50 arm (Figure 6 and Additional file 7: Table S5) of the
predicted hairpins. However, one miRNA, tgo-novel-1,
the miRNA:miRNA* ratios showed strain-specific
pattern. The reads from 30 arm were dominant in ME49
(50/30 read ratio: 443/6212), whereas in RH, the reads
number from 50 arm is higher (50/30 read ratio: 9632/
2838) (Additional file 7: Table S5).
Validation of miRNAs expression by Northern blot
Five novel miRNAs (tgo-novel-1-1-3p, tgo-novel-12-1,
tgo-novel-40, tgo-novel-41, and tgo-novel-14-2) with
relatively high abundance identified by sequencing
were verified by Northern blot. Specific hybridization
with probes of three miRNAs was observed at 23 nt
(Figure 7). In addition, all probes showed hybridization
signals to the pre-miRNA transcripts of about 80 nt,
except for the precursor of tgo-novel-40, whose signal
was detected at 100 nt. Further, the hybridization
signal to the mature miRNAs of tgo-novel-40,
tgo-novel41 and their pre-miRNAs was more intense in ME49
than that in RH, which implying higher expression level
in ME49. The expression of tgo-novel-1-1-3p was only
detected in RH, which was similar with the results of
sequencing analysis. On the contrary, the mature miRNA
of tgo-novel-14-2 was only detected in ME49, though
the pre-miRNAs with a similar expression level detected
in the two strains. The inconsistency with the sequencing
data might be due to the slow processing of pre-miRNAs
in RH strain. No hybridization was seen with any probe
to the mouse miRNAs.
Discussion
MiRNAs are recognized as critical regulators in gene
expression at the post-transcriptional level. Previous
studies have found that T. gondii possesses a complete
RNA silencing pathway which suggests that small
noncoding RNAs may play a critical role in the parasite
development and its parasitization in the hosts [30]. In
this study, the profiles of small RNA populations of the
two distinct strains of T. gondii were investigated.
The distribution of small RNAs in the two small RNA
libraries generated after deep sequencing was compared.
A predominant number of small ncRNAs was
strainFigure 6 The sequence and the secondary stem-loop structure of tgo-novel-12-14 identified in T. gondii. Sequences and the number of
reads of the mature miRNA and the complementary miR* are represented in red and blue respectively. The predicted structure of the pre-miRNA
is represented on the right side.
Figure 7 Characterization of four miRNAs by Northern-blot.
Lanes from left to right are RNAs from ME49 strain (ME49), RH strain
(RH) and a mouse. Total RNA isolation from ME49, RH strain and a
mouse were visualized by ethidium bromide staining and served
as loading controls at the top panel. LNA probes corresponding to
tgo-Novel-1-1-3p, tgo-Novel-12-1, tgo-Novel-40, tgo-Novel-41,
tgo-Novel-14-2 were used. Probe tgo-Novel-1-1-3p and tgo-Novel-40
only hybridized to the RNAs of RH strain and ME49, respectively.
While probes of tgo-Novel-12-1, tgo-Novel-41 and tgo-Novel-14-2
hybridized to RNA of both strains. More hybridization was seen with
tgo-Novel-41 probe to ME49. More hybridization was seen with
pre-miRNAs except with the probe of tgo-Novel-41. No hybridization
was detected with mouse RNA with any probe. Hybridization to
mature miRNAs was marked with asterisks.
specific (Figure 1 and Figure 2) and the strain-specific
small ncRNA including miRNAs were more in RH strain
than in ME49 strain (Figure 1). The difference in
miRNA numbers found in the two libraries could be due
to the less presentation of ME49 genomic sequences in
the databases which may affect small RNA identification.
Further, there was a clear difference in the tendency of
the length distribution in small RNAs between the two
strains. In RH, the 26 nt RNAs represented the
predominant species (Figure 3, Additional file 5: Table S3), while
in ME49, about 18.92% of the total small RNAs were
21 nt in size, which was the most abundant class. This
might be due to the genetic differences between the two
parasite strains, which also suggested that the two
parasite strains are biologically different. Previous study
reported that the structural features of pre-miRNA
hairpins might influence the efficiency of Dicer binding and
specificity of precursor cleavage, which leads to the
length diversity of miRNAs [46,47]. The reasons of
strain-specific length distribution of the small ncRNAs
between the two strains of T. gondii remain vague,
further studies are needed to dissect the mechanism in
sncRNA processing that may be associated with
strainspecific gene regulation.
In total, we identified 17 conserved miRNA and 339
species-specific miRNAs in the two strains of T. gondii,
of which 7 miRNAs sequences were reported by Braun
et al. in 2010 [30]. Interestingly, about 5% of T. gondii
miRNAs were categorized as conserved and accounted
for less than 1% of the read counts, whereas more
than 99% of the remaining miRNAs were recognized as
species-specific. This observation supports the earlier
finding that T. gondii possesses a RNA-associated gene
regulation machinery which is phylogenetically diverged
from mammals but more similar to plants [30]. Further,
let-7 and lin-4, the two most conserved miRNAs in
metazoan, were not found in T. gondii indicting that
the fine-tuning mechanism of miRNAs in T. gondii was
distinct from other species.
During the process of the biosynthesis of miRNAs,
miRNA and miRNA* (or miR*) were generated by
enzymatic cleavage of the 7080 nt precursor hairpin.
The functional strand (miRNA) of the small RNA duplex
is preferentially loaded into the RISC as the guide strand,
while the other strand, the passenger strand (miRNA*),
is degraded [44,45]. However, recent studies suggested
that mature miRNAs can be generated from both
strands of the pre-miRNA duplex [48-50]. In C. elegans
and related nematodes, it has been reported that the
diversity of miRNAs was, at least partially, due to the
arm-switching and hairpin shifting [51]. We found that
tgo-novel-1 changed the miRNA strand with arm
switching of the same hairpin between the two strains of
T. gondii. In RH strain, the dominant miRNA of
tgonovel-1 was derived from the 30 arm, while in ME49 it
seemed that mature miRNA was only derived from the
50 arm. Due to the fact that the sequences of miRNAs
derived from the two arms of the same hairpin were
complementary, they likely regulate different target
sequences. Further dissection of the function of the
miRNAs derived from the two arms of the same hairpin
might lead to deep understanding of the parasite biology.
Conclusion
In summary, 17 conserved miRNAs related to 2
metazoan miRNA families and 339 novel miRNAs were
identified in the two genetically different strains of T. gondii.
The majority of miRNAs were species-specific, which
supports the finding that T. gondii is an evolutionarily
diverged organism from other protozoana. The
difference in expression abundance of certain miRNAs as well
as the arm-switching in pre-miRNA processing leading
to different miRNA species in the two parasite strains
suggested that there was a fine-tuning mechanism of
miRNA biogenesis in distinct strains of T. gondii.
Understanding the genetic factors that regulate T. gondii gene
expression could contribute to the development of
specific tools to control the transmission of the parasite.
Additional file 1: Figure S1. The flow chart for detailed analysis of
small RNAs isolated from the two strains of T. gondii.
Additional file 2: Table S1. General information of the two libraries.
Description: This file contains summary data from high-throughput
sequencing of the two small RNA libraries.
Additional file 3: Table S2. Small RNA classification. Description: This
file contains the reads of all small RNA transcripts identified and their
relative portions in the library.
Additional file 4: Figure S2. Statistic analysis of sncRNAs identified in
the two libraries. The reads at unique and total levels of the small RNAs
in different lengths ranged from 18 to 30 nt were plotted. The difference
between the lengths at unique level was significant (p < 0.0001).
Additional file 5: Table S3. Length distribution of small RNAs identified
in the two strains of T. gondii. Description: This file contains the reads of
small RNAs with different lengths and their relative portions in the library.
Additional file 6: Table S4. Conserved (common) miRNAs and the
genomic loci of the encoding genes identified in the two strains of T.
gondii. Description: miRNAs with the same sequences could be derived
from pre-miRNAs (the sequences may not be completely the same)
located at different genomic loci. Their names are indicated with an
additional dash-number suffix.
Additional file 7: Table S5. Novel (unique) miRNAs and the genomic
loci of the encoding genes identified in distinct strains of T. gondii.
Additonal file 8: Table S6. Comparative analysis of the expression of
novel miRNAs in the two libraries analyzed by software IDEG6 and Edger
respectively.