Whole-transcriptome profiling of bacterial samples can provide significant insights into mechanisms of prokaryotic metabolism. Unlike DNA profiling, it can also potentially discriminate between live and dead organisms in a mixed population, since RNA molecules have a shorter half-life than DNA molecules. Here, we describe how the Ion Torrent™ platform can be used to profile the transcriptome from E.coli and S.aureus cultures. The Ion Total RNA-Seq kit and AB Library Builder™ were used for semi-automated library synthesis from Ribo-depleted RNA. Using the Partek Flow Pipeline for Ion Whole Transcriptome Analysis, we performed our alignment and analysis. We obtained between of 18-29 million reads per sample, allowing average coverage depth of around 500x for E.coli and around 1000x for S.aureus. Correlations of expression levels between replicates was excellent, with a Pearson Correlation Coefficient averaging greater than 0.97. between replicate samples. The top quartile of expressors had Pearson Correlation Coefficients of greater than 0.99 for both E.coli and S.aureus.These data demonstrate that the Ion Torrent Proton system provides an ideal workflow and capacity for sequencing and analysis of prokaryotic transcriptomes.

1. A. B. C.
D. E. F.
Chaitali Parikh, Dalia Dhingra
Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080
MATERIALS & METHODS
ABSTRACT
Whole-transcriptome profiling of bacterial samples can provide significant insights
into mechanisms of prokaryotic metabolism. Unlike DNA profiling, it can also
potentially discriminate between live and dead organisms in a mixed population,
since RNA molecules have a shorter half-life than DNA molecules. Here, we
describe how the Ion Torrent™ platform can be used to profile the transcriptome
from E.coli and S.aureus cultures.
The Ion Total RNA-Seq kit and AB Library Builder™ were used for semi-automated
library synthesis from Ribo-depleted RNA. Using the Partek Flow Pipeline for Ion
Whole Transcriptome Analysis, we performed our alignment and analysis. We
obtained between of 18-29 million reads per sample, allowing average coverage
depth of around 500x for E.coli and around 1000x for S.aureus. Correlations of
expression levels between replicates was excellent, with a Pearson Correlation
Coefficient averaging greater than 0.97. between replicate samples. The top
quartile of expressors had Pearson Correlation Coefficients of greater than 0.99 for
both E.coli and S.aureus.These data demonstrate that the Ion Torrent Proton
system provides an ideal workflow and capacity for sequencing and analysis of
prokaryotic transcriptomes.
CONCLUSIONS
The Ion Torrent Proton™ system provides an automated workflow for generating highly
reproducible data that simplifies prokaryotic transcriptome research.
For Research Use Only. Not for use in diagnostic procedures.
REFERENCES
1.For more information, please visit www.lifetechnologies.com.
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property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
Partek and Flow are trademarks of Partek Incorporated.
Bacterial transcriptome profiling using Ion Torrent Proton technology
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com
RESULTS
Workflow Steps Done on ABLB Instrument
2 Hrs 1-2 Hrs 3 Hrs
~70-85% of total sample prep
workflow (~6-7 hrs) can be
automated by the AB Library
Builder
AB Library
Builder™
With Ion
Total RNA-
Seq kit
Size
Select
Purify
RNA
Ribo
-depletion
Frag &
Purify
RNA
Hyb & Lig
adapters
Reverse
transcribe
PCR &
Clean-up
Sequence
AB Library
Builder™
With
iPrep RNA
kit
A. B. C. D.
Figure 1. Total RNA Library workflow on AB Library Builder ™ System: Fast and
Simple
(A) RNA purification can be performed in two hours with the AB Library Builder ™ using the
iPrep RNA kit. (B) After ribo-depletion and fragmentation, automated library preparation for
an Ion Torrent platform can be done in 3 hours using AB Library Builder ™ with the Total
RNA-Seq kit. (C) These libraries can then be amplified for templating on the Ion One Touch
2 System. (D) Sequencing is performed with the Ion Proton System.
Figure 2. Partek Flow for Whole Transcriptome Analysis.
Partek Flow WT Pipeline for Ion Torrent was used alignment and analysis. Using the
PartekFlowUploader plugin, the unmapped reads were automatically transferred into Partek
Flow. The Partek Flow WT Pipeline for Ion Torrent was then started that includes the two-
step alignment method for Ion Proton™ transcriptome data (Tophat2 and Bowtie2) followed
by quantification and differential gene expression analysis.
Figure 3. ERCC Analysis
Output from the ERCC_Analysis plugin in Torrent Suite is shown, a logarithmic graph of the
ERCC concentration versus the ERCC counts from the transcriptome sequencing data with
an R-squared of 0.94. The R-squared ranged from 0.90 up to 0.96 for all the samples with 3
of the samples at 0.96. The ERCCs comprised of 1.19% of total reads for this sample. They
ranged from 0.80% up to 2.22% for all samples. The colors of the data points reflect the map
quality with the majority of reads having greater than 80 for the ERCCs above the threshold
count of 10.
A. B. C.
Figure 4. Example a whole transcriptome sequencing run with E. .coli
(A) Heat map of an Ion Proton PIv2 chip indicating 95% loading of the wells with ISPs. (B)
After filtering out low quality and polyclonal reads, there were close to 80 million reads. (C)
Read length histogram after quality trimming demonstrated median read length of 110 base
pairs.
total reads aligned (%) unique (%) coverage (%)
average coverage
depth
average
length
average
quality
average
mapping quality GC (%)
E. coli
BC3 18,569,344 98.04 96.39 95.36
376.16
(SD 3,282.17) 102.34 23.81 45.99 (SD 10.94) 51.23
BC4 26,008,453 97.84 96.59 94.01
636.28
(SD 7,113.69) 123.43 23.28 45.78 (SD 10.34) 51.89
BC5 20,678,168 97.94 96.54 94.52
489.07
(SD 5,189.36) 119.04 23.36 45.89 (SD 10.52) 51.75
Staph
aureus
BC1 23,600,051 94.80 93.80 87.41
976.79
(SD 22,791.1) 111.97 23.29 44.33 (SD 12.25) 38.83
BC2 27,563,733 96.15 94.92 96.28
1,118.51
(SD 17,078.3) 122.98 23.00 41.77 (SD 14.54) 37.12
BC6 29,589,992 94.87 92.35 89.61
1,301.93
(SD 30,244.2) 116.93 23.34 41.63 (SD 15.88) 39.51
Table 1. Alignment statistics.
The combined alignments from a two-step alignment method (TopHat2 and Bowtie2) automated in the
Partek Flow WT Pipeline for Ion Torrent. Default parameters for the pipeline was used for all alignments. Over
94% of all reads aligned with over 92% of all reads aligning uniquely.
E. Coli Staph aureus
BC3 BC4 BC5 BC1 BC2 BC6
rRNA (%) 0.077 0.031 0.059 0.340 0.149 1.347
5S rRNA (%) 0.005 0.002 0.004 0.023 0.012 0.136
16S rRNA (%) 0.007 0.025 0.021 0.106 0.046 0.440
23S rRNA (%) 0.008 0.026 0.017 0.210 0.091 0.771
non-ribosomal reads
from 3-plex PI chip 18,190,630 25,439,651 20,240,890 22,296,402 26,462,369 27,695,202
Table 2. Percent ribosomal RNA reads after ribo-depletion.
The percent reads classified as rRNA and separated into 5S rRNA, 16S rRNA, and 23S rRNA are shown here as
the percent of reads for each E. coli and Staph aureus sample. All samples had over 18 million reads that were not
rRNA when 3 samples were sequenced on a single Ion Proton PIv2 chip.
E. coli BC3 BC4 BC5
BC3
0.9777 0.9865
0.9891 0.9934
0.7305 0.6783
BC4
0.9967
0.9981
0.6781
Staph aureus BC1 BC2 BC6
BC1
0.9918 0.9993
0.9924 0.9993
0.7699 0.7046
BC2
0.9932
0.9936
0.7928
All genes
Top quartile (Highest expressed genes)
Bottom quartile (Lowest expressed genes)
A. B.
Table 3. Pearson correlation coefficients.
(A) Pearson correlation coefficients were determined between the E. coli technical replicates for all
genes, the top quartile of expressed genes and the bottom quartile of expressed genes. For all genes
and the top quartile, the Pearson correlation coefficient was over 0.97. For the bottom quartile of
expressed genes, the Pearson correlation coefficient was lower, as expected, but still higher than 0.67.
(B) Pearson correlation coefficients were determined between the Staph aureus technical replicates for
all genes, the top quartile of expressed genes and the bottom quartile of expressed genes. For all genes
and the top quartile, the Pearson correlation coefficient was over 0.99. For the bottom quartile of
expressed genes, the Pearson correlation coefficient was lower, as expected, but still higher than 0.70.
E. Coli
Staphylococcus Aureus
Figure 5.
High degree of overlap in the E. coli samples for genes that are the (A) top 20 expressors, (B) the top
quartile expressors , and (C) the bottom quartile expressors. High degree of overlap in the staph aureus
samples for genes that are the (D) top 20 expressors, (E) the top quartile expressors , and (E) the
bottom quartile expressors.