APPLICATION NOTE SeqStudio Genetic Analyzer

The 16S Direct workflow

Microbial identification using 16S gene sequencing on the SeqStudio
Genetic Analyzer and analysis with MicrobeBridge software

In this application note we present:

• A fast and economical workflow called “16S Direct” for
bacterial identification at the species level by PCR and
Sanger sequencing of the 16S rRNA gene

• The use of the Applied Biosystems™ BigDye™ Direct kit

for high-resolution Sanger sequencing

• The use of the innovative Applied Biosystems™

SeqStudio™ Genetic Analyzer for capillary electrophoresis

• The use of the freely available Applied Biosystems™ or laboratories may not need high-powered solutions.
MicrobeBridge™ software with link to the US Centers for The recently introduced Applied Biosystems™ SeqStudio™
Disease Control and Prevention (CDC) and MicrobeNet Genetic Analyzer provides a low-cost alternative to
for data analysis high-throughput instruments.

Introduction In this application note, we present a novel, fast, and

Rapid and accurate identification of infectious, fastidious, economical 16S gene sequencing workflow called
or noncultivable bacteria is a major challenge for clinical “16S Direct” that is optimized for use on the SeqStudio
practice and public health surveillance. Based on the Genetic Analyzer.
groundbreaking phylogenetic research work of Woese et
al. [1] and others, sequencing of the 16S ribosomal RNA SeqStudio Genetic Analyzer and 16S Direct workflow
(rRNA) gene has emerged as the preferred method for The SeqStudio Genetic Analyzer is an affordable capillary
taxonomic classification and identification of bacteria. electrophoresis (CE) system that can perform both
Today, many products and literature reviews demonstrate automated Sanger DNA sequencing and high-precision
the use of sequencing of the 16S gene as the gold sizing and analysis of multicolor fluorescent DNA
standard for identification at the species level (for reviews, fragments. The instrument can accommodate a
see references 2 and 3). There are numerous protocols 96-well plate loaded with sequencing reactions that are
and a published guideline [4] for PCR and sequencing of sequentially electrophoresed in batches of four samples
the 16S gene for research and applied use. In addition, per run. The sequencing samples can be processed in
several databases from public institutions and commercial a 30 min “short” run cycle, resulting in DNA sequences
companies are available for comparing 16S sequences of about 500 bases each. This is sufficient to reconstruct
with typed or annotated entries. the almost complete sequence (~1.5 kb) of the 16S gene
and thus identify the species of a particular sample in only
The Applied Biosystems™ MicroSEQ™ 16S gene PCR 4 sequencing reactions and 1 run.
sequencing kit and MicroSEQ™ database have long been
trusted for microbial identification in pharmaceutical
production settings. Although the MicroSEQ system works
well on high-throughput instruments, some investigators
The procedure described here can be adapted to individual The basic idea behind the 16S Direct workflow is to reduce
needs, such as different primer designs or running the the number of sequencing reactions to 4 in order to fully
sequencing reactions on a CE instrument such as an use the running capacity of 4 capillaries on the SeqStudio
Applied Biosystems™ 3500 series genetic analyzer. Other instrument. This maximizes the throughput of samples
established or commercial 16S sequencing methods and within a given time window.
protocols using Applied Biosystems™ BigDye™ sequencing
chemistry will also work on the SeqStudio instrument, but The 16S Direct workflow
may require more steps and time to prepare additional CE An overview and an example timeline of the 16S Direct
runs as more sequencing reactions for assembling the workflow are shown in Figure 1 and in more detail in Figure
complete gene sequence are needed (Table 1). 2. Starting in the morning, individual bacterial colonies
are picked from agar culture plates and PCR-ready DNA
Table 1. Number of PCR reactions, sequencing reactions, and runs is extracted using, for example, the Applied Biosystems™
on the SeqStudio Genetic Analyzer (30 min each) required to identify PrepMan™ Ultra Sample Preparation Reagent. PCR
a number of bacteria after 4 hr CE time (arbitrary time point). reactions “A” and “B” are then set up followed by a quick
No. of CE runs
No. of needed on No. of bacteria
check for quantity and expected size of the two amplicons
No. of PCR sequencing SeqStudio identified after 4 hr by standard agarose or Invitrogen™ E-Gel™ agarose gels.
reactions reactions instrument (i.e., after 8 CE runs)
A total of 4 sequencing reactions are then set up using
16S Direct
2 4 1 8
workflow the BigDye Direct sequencing reagent and cycled. The
MicroSEQ reactions are then purified with Applied Biosystems™
Full Gene
16S rDNA
3 6 2 5 BigDye™ XTerminator™ reagent, and the samples are
Sequencing Kit electrophoresed on the SeqStudio Genetic Analyzer in the
Traditional early afternoon. The instrument continuously generates a
lab-developed 1 8 2 4
protocol data set of 4 sequences every 30 min when operating in
short-run mode. The sequence files are then immediately
available for secondary data analysis using MicrobeBridge
software and BLAST™ alignment with a microbial database.

11:15 a.m.–12:30 p.m.

Cycle sequencing

9:15 a.m.–11 a.m. 12:30 p.m.–1 p.m.

PCR including analysis CE preparation using
with E-Gel agarose gel BigDye XTerminator reagent

1:45 p.m.: First CE run is completed for sample 1;

first data set is available for assembly and
BLAST alignment
2:15 p.m.: Second run is completed for sample 2;
second data set is available for assembly and
1:15 p.m. onwards BLAST alignment
8 a.m.–9 a.m.
DNA prep CE on SeqStudio instrument 2:45 p.m.: Third run is completed for sample 3;
third data set is available for assembly and
BLAST alignment
3:15 p.m.: Fourth run is completed for sample 4;
fourth data set is available for assembly and
BLAST alignment …
… and so on
Sequencing data are uploaded around-the-clock
to cloud or server after each run is completed and
are available for remote analysis

Figure 1. The 16S Direct workflow—an example time line.

 • Prepare gDNA from bacterial isolate
DNA • Use your preferred or verified method to prepare PCR-ready DNA

• Set up PCR reactions “A” and “B” with BigDye Direct kit (as described in “Materials Time (manual): 2–10 min
PCR and methods” section) Time (on PCR cycler): 60 min

Time (manual): 2–5 min

E-Gel agarose • Check PCR success with an E-Gel or other agarose gel
Time (on device): 10–15 min
gel

• Set up forward and reverse sequencing reactions with amplicons “A” and “B” using the Time (manual): 2–10 min
Sequencing BigDye Direct sequencing kit Time (on PCR cycler): 70 min

BigDye • Purify sequencing reaction with BigDye XTerminator reagent Time (manual): 2–5 min
XTerminator • Transfer supernatant to CE plate Time (on device): 20–30 min
reagent
• Set up CE run on the SeqStudio Genetic Analyzer Time (manual): 1–5 min
• Short run (each run of 4 sequencing samples = 1 specimen) or Time (on instrument): 30 min
CE • Long run (each run of 4 sequencing samples = 1 specimen) Time (on instrument): 100 min

• Assemble sequencing files with MicrobeBridge software Time (manual): 1–10 min
MicrobeBridge
• Copy into PC memory
software

• Paste into preferred 16S database (e.g., NCBI microbial genomes or CDC MicrobeNet) Time (manual): 1–2 min
BLAST
• Review or verify top hit Time (intellectual): 1–5 min
alignment

Figure 2. Steps of the 16S Direct workflow.

Materials and methods PCR set “A” amplifies the 5� half of the 16S rRNA gene from
DNA position 8 to 802, and PCR set “B” amplifies the 3� half of
The genomic DNA (gDNA) used for this research was the 16S rRNA gene from position 785 to 1,511 (Figure 3).
from bacterial strains of known species obtained from Forward PCR primers are modified at the 5� end with the
ATCC and diluted to 10 ng/μL in Invitrogen™ TE buffer M13 forward primer sequence and likewise, reverse PCR
(Tris 10 mM, EDTA 0.1 mM). To prepare PCR-ready DNA primers with the M13 reverse primer sequence. M13 primer
from bacteria growing on an agar plate or other biological sequences are shown in lowercase letters. Note that the
or environmental matrix, the PrepMan Ultra Sample internal 16S gene–specific primers are complementary
Preparation Reagent is a suitable product that is fast, (Figure 3) and thus form a short overlapping segment of
economical, and easy to use. Other options are described 18 bp that can be used to link segments “A” and “B” for
at thermofisher.com/gdna. full gene sequence assembly using the MicrobeBridge
software. The advantage of this particular primer design
PCR is that only 4 sequencing reactions are required for near
For PCR of the 16S rRNA gene, the following primers were full-length coverage of the 16S gene, which matches the
custom ordered from thermofisher.com/oligos: 4-capillary capacity of the SeqStudio instrument for a
single run.
16S_A_FWD (F8): 5�-tgtaaaacgacggccagtAGAGTTTGATC-
MTGGCTCAG-3� For PCR, 5 μL of the BigDye Direct PCR reagent was
16S_A_REV (R802): 5�-caggaaacagctatgaccTACCAGGG- combined with 3.5 μL water and 0.5 μL primer pair “A”
TATCTAATCC-3� or “B” (each primer at 10 μM) and 1 μL DNA (typically
16S_B_FWD (F785): 5�-tgtaaaacgacggccagtGGATT- 1–10 ng) and amplified in an Applied Biosystems™ ProFlex™
AGATACCCTGGTA-3� PCR System using these cycling conditions: initial hot
16S_B_REV (R1511): 5�-caggaaacagctatgaccCGGT-
start at 95°C for 5 min followed by 8 cycles at 95°C for
TACCTTGTTACGACTT-3�
15 sec, 50°C for 15 sec, and 68°C for 90 sec, followed by
27 cycles at 95°C for 15 sec, 65°C for 15 sec, and 72°C
for 90 sec.
 A
V V V V V V V V V
1 2 3 4 5 6 7 8 9

B 8
M13F

Amplicon A
M13R
802

785
M13F

Amplicon B
C 8
M13R
1511
M13F
M13R
802
785
M13F
M13R
1511

Figure 3. Sequencing primer-binding sites. Schematic representation of the 16S rRNA gene showing (A) variable regions (V1–V9) with high sequence
diversity between species, and general and conserved regions (white segments) that are targets for the M13-tagged primers. The segment between
nucleotides 785–802 where the sequences overlap serves as a primer-binding site for the (B) PCR amplicons “A” and “B”. (C) The M13 tags are used as
sequencing primer-binding sites.

After PCR, 15 μL of low-EDTA TE buffer was added to the sequencing reagent, and unique M13 sequencing primers
reaction and 2.5 μL (~10%) was used for electrophoresis on that bind to the M13 forward and reverse sequences
a 2% E-Gel agarose gel to verify the presence of amplicon present in the PCR amplicon. The BigDye Direct reagent
“A” (~830 bp) or amplicon “B” (~750 bp) as a clearly visible provides high resolution of the nucleotide sequences
single band in an estimated amount of 20–50 ng. close to the 5� end and does not require a separate PCR
primer purification step, thereby saving time and extra
High-resolution Sanger sequencing with the BigDye reagents. A sample setup of 4 cycle sequencing reactions
Direct sequencing reagent for sequencing M13-tagged PCR amplicons “A” and “B” in
The BigDye Direct kit is comprehensive and economical, both directions is shown in Table 2.
containing PCR reagent (see above), BigDye Direct

Table 2. Setting up the forward and reverse BigDye Direct sequencing reactions. Note that the Applied Biosystems™ BigDye™ 5X sequencing buffer
is not included in the BigDye Direct kit and must be obtained separately. The 5X buffer is part of the standard BigDye sequencing v1.1 or v3.1 kits or can
be purchased as a stand-alone item.
Volume for 2 forward sequencing Volume for 2 reverse sequencing
Reagent reactions (1 specimen) reactions (1 specimen)
Molecular biology–grade water (not included in BigDye Direct kit) 12 µL 12 µL
BigDye v1.1 and v3.1 sequencing buffer 5X (not included in the BigDye Direct kit) 3 µL 3 µL
BigDye Direct M13 forward primer (included in the BigDye Direct kit) 1 µL –
BigDye Direct M13 reverse primer (included in the BigDye Direct kit) – 1 µL
BigDye Direct sequencing reagent (included in the BigDye Direct kit) 2 µL 2 µL
Total volume 18 µL (= 2 x 9 µL) 18 µL (= 2 x 9 µL)
 
9 µL forward sequencing reaction + 1 µ
L
Well or tube 1
amplicon A
9 µL reverse sequencing reaction + 1 µ
L
Well or tube 2
amplicon A
9 µL forward sequencing reaction + 1 µL
Well or tube 3
amplicon B
9 µL reverse sequencing reaction + 1 µ
L
Well or tube 4
amplicon B
Cycle sequencing on a ProFlex PCR System was After completion of a CE run cycle, the 4 sequencing files
performed using this profile: 1 cycle comprised of 37°C for PCR segments “A” and “B” are immediately available for
for 15 min, 80°C for 2 min, and 96°C for 1 min; 13 cycles data analysis and also remotely retrievable through internet
at 96°C for 10 sec, 50°C for 5 sec, and 60°C for 1 min cloud connectivity. This helps enable urgent identification
15 sec; 6 cycles at 96°C for 10 sec, 50°C for 5 sec, analysis away from the lab or after lab hours.
and 60°C for 1 min 30 sec; 6 cycles at 96°C for 10 sec,
50°C for 5 sec, and 60°C for 2 min 30 sec. After cycle Data analysis
sequencing, 50 μL of BigDye XTerminator suspension was The output files from the SeqStudio Genetic Analyzer are
added to each sequencing reaction. It consisted of 1 part sequencing files in .ab1 format, which can be viewed in
BigDye Xterminator beads (10 µL/sample) and 4 parts of electropherogram trace mode or in text mode. In order
SAM Solution (40 μL/sample) and was prepared in bulk to reconstitute the entire sequence of the 16S rRNA
for all samples shortly before use. The sequencing plate gene from both strands of amplicons “A” and “B”, the
with the BigDye XTerminator suspension–treated samples .ab1 files need to be assembled, i.e., an overlap needs to
was vigorously vortexed for 30 min and then centrifuged be established and a contiguous consensus sequence
for 2 min to pellet the beads; 20 μL of the supernatant was (“contig”) needs to be generated. This can be conveniently
transferred to a fresh plate, centrifuged for 1 min to remove done using the MicrobeBridge v.1.1 software, which
any residual air bubbles, and then placed on the SeqStudio is available as a free download at thermofisher.com/
Genetic Analyzer for CE. order/catalog/product/A29238. Briefly, the workflow is
as follows:
Sequencing by CE on the SeqStudio
1. Import and organize the 4 associated sequence files
Genetic Analyzer
(i.e., forward and reverse for amplicon “A” and “B”) for
The samples can be run either as short (30 min), medium
each specimen.
(45 min), or long runs (105 min) using BigDye Direct
chemistry and a regular sequencing run module (note: do 2. Click on the “Review” tab, which initiates base calling
not use a BigDye XTerminator run module). Long runs yield and the assembly process that results in an overview
nearly complete bidirectional coverage, whereas short runs of the sequence assembly (Figure 4), details of the
yield approximately complete, 40% bidirectional coverage. traces (Figure 5), and quality metrics.
Complete and accurate assembly of near full-length 16S
gene (~1,450 bases) sequences can be reliably achieved
with high-quality (quality value (QV) >35) short-run data.

Figure 4. Overview of the assembly of the 4 sequencing traces: forward and reverse for amplicon “A” and forward and reverse for amplicon “B”.
Figure 5. Trace view of the junction between amplicons “A” and “B”.

3. Review the assembly output, which enables correcting and useful public service is provided by the CDC at
any miscalls in the consensus sequence. Ambiguities https://microbenet.cdc.gov/.
in the assembly are indicated by a red bar (Figure 4),
which can be inspected and edited in the rare event Adjusting the trimming stringency to facilitate
that it is necessary. The MicrobeBridge assembler alignment and contig assembly
usually determines the consensus base call by the When the overlap in the center between amplicons “A”
sequence trace with the highest quality and thus the and “B” is shorter than 10 nucleotides, the alignment fails
lowest probability of error. Note the correct orientation and a scrambled assembly with a cluster of red bars will
of the forward and reverse traces for amplicons “A” and occur. This undesired outcome can often be rectified by
“B” (Figure 4) and verify that the overall length of the adjusting (i.e., relaxing) the “clear range” trim parameters
assembled sequence reaches ~1,450 bases. in the project analysis settings (Figure 7). By default, the
clear range is set to 3 bases, which is fairly stringent and
4. When the assembly results look satisfactory, open sufficient to generate a consensus sequence between the
the “Contig review” tab and click “Copy contig” amplicon “A” forward and “A” reverse strands or likewise
(Figure 6A). This will copy the assembled sequence amplicon “B” forward and “B” reverse strands. Due to the
onto the memory clipboard for subsequent pasting relatively short sequence overlap between the 3� end of
into a BLAST alignment of personal preference. segment “A” and the 5� end of segment “B”, the default
For feasibility of demonstration in this application trimming can sometimes undercut this limit when the
note, we have used the NCBI Microbial Nucleotide sequence quality of the terminal bases is not ideal. In this
BLAST tool, which compares a query sequence to case, the alignment can be achieved by setting the “clear
fully sequenced microbial genomes for identification range” to a higher value (e.g., 4, 5, 6) until the alignment
(Figure 6B and C). For additional information, is successful. In rare cases, an alignment and assembly
please refer to https://blast.ncbi.nlm.nih.gov/ between amplicons “A” and “B” may not be achieved.
Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_ In such cases, the species identification by nucleotide
SPEC=MicrobialGenomes. alignment has to be done separately with amplicons “A”
and “B”, which should produce the same result. The
The “Open MicrobeNet” button (Figure 6A) only difference with an alignment with a joint “A + B”
provides a link to the CDC MicrobeNet portal for segment is that the total number of aligned nucleotides
pasting the query sequence into the CDC localized will be 15–20 nt shorter (i.e., missing the overlap segment
alignment tool for identification and further detailed between “A” and “B” consisting of the primer region
microbiological information on the lead hit. This free F785–R802 for which the sequence is known).
 A

A CDC Virtual Reference Laboratory

B C

Figure 6. Data analysis workflow. (A) The “Copy contig” button (circled in red) copies the assembled consensus sequence onto a memory clipboard
ready for pasting into either (B) the NCBI Microbial Nucleotide BLAST portal or any other search application. For identification, the top hit of the results is
selected and (C) the species ID is shown with alignment and metrics (e.g., % identity). Alternatively, using the “Open MicrobeNet” button (6A ), the contig
can be pasted into the CDC MicrobeNet BLAST tool for identification and further information on the species.

Results
Long sequencing runs
To demonstrate the feasibility of the primer design and
workflow described above, we have processed DNA
derived from 6 representative microbial organisms
belonging to 5 different phyla: Bacteroidetes, Fusobacteria,
Proteobacteria, Actinobacteria, Firmicutes, and
Verrucomicrobia. Table 3 shows a summary of the
long-read sequencing results from the SeqStudio Genetic
Analyzer. Sequencing metrics for each of the 4 sequencing
reactions are listed.

The quartet of sequencing files was analyzed by

Figure 7. Accessing the project analysis settings. The project analysis MicrobeBridge software in 3 ways:
settings can be accessed by clicking the “Settings” button. The stringency
of the base trimming can be relaxed by increasing the default value from 1. Consensus of forward and reverse reads of PCR
“3” to “4”, “5”, “6”, or “7”.
amplicon “A” (sequencing reactions 1 and 2)

2. Consensus of forward and reverse reads of PCR

amplicon “B” (sequencing reactions 3 and 4)

3. Consensus of forward and reverse reads of PCR

segments “A and B” (sequencing reactions 1, 2, 3,
and 4).
Table 3. Summary of long CE runsequence data metrics, alignment results, and identification of the microbial DNA test panel representing
5 bacterial phyla. “Sample score” is a consolidated quality measure: the target is a value of 35 or higher. CRL is contiguous read length in nucleotides;
QV20 is the longest number of nucleotides with a minimum quality value (QV) of 20 (i.e., probability of error of 1 in 100); RFU is the average signal strength
of all 4 dye traces in relative fluorescent units. The MicrobeBridge software trim setting is the value set as the clear range for trimming end bases in the
analysis protocol (Figure 7). Top ID is the first and highest-ranking listing in the result table of the BLAST search.

Sequencing trace metrics MicrobeBridge software BLAST results

Sample number, PCR Sequencing Sequencing SeqStudio Sample Trim setting for No. of nt No. of
organism of DNA input primer set primer trace No. run mode score CRL QV20+ Signal RFU contig assembly Top ID Identity (%) aligned gaps

1 Forward 1 Long sequence 49 772 756 1,388

A
Reverse 2 Long sequence 47 783 780 1,099
} 3 (default) Bacteroides fragilis 99.87% 756 0

Bacteroides fragilis
ATCC 25285D-5
Forward 3 Long sequence 39 717 703 710
B
Reverse 4 Long sequence 43 711 705 688
} 3 (default) Bacteroides fragilis 99.56% 677 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 5 Bacteroides fragilis 99.72% 1,451 0

2 Forward 1 Long sequence 47 760 761 1,012

A
Reverse 2 Long sequence 47 761 749 763
} 3 (default) Fusobacterium nucleatum 99.73% 734 0

Fusobacterium nucleatum
ATCC 25586D-5
Forward 3 Long sequence 43 720 710 782
B
Reverse 4 Long sequence 48 729 724 551
} 3 (default) Fusobacterium nucleatum 100% 690 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 3 (default) Fusobacterium nucleatum 99.86% 1,442 0

3 Forward 1 Long sequence 34 53 329 1,088

A
Reverse 2 Long sequence 47 787 777 975
} 3 (default) Acinetobacter baumannii 99.73% 749 0

Acinetobacter baumannii
ATCC 17978D-5

}
Forward 3 Long sequence 43 724 717 877
B 3 (default) Acinetobacter baumannii 100% 689 0
Reverse 4 Long sequence 50 697 708 533

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 3 (default) Acinetobacter baumannii 99.86% 1,456 0

4 Forward 1 Long sequence 34 54 133 762

A
Reverse 2 Long sequence 47 806 794 656
} 3 (default) Actinomyces oris 97.80% 773 6

Actinomyces naeslundii
ATCC 12104D-5
Forward 3 Long sequence 43 722 710 942
B
Reverse 4 Long sequence 49 720 718 617
} 3 (default) Actinomyces oris 99.28% 692 2

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 3 (default) Actinomyces oris 98.52% 1,483 3

4 Forward 1 Long sequence 34 54 133 762

A
Reverse 2 Long sequence 47 806 794 656
} 3 (default) Actinomyces naeslundii 100% 773 0

Actinomyces naeslundii
ATCC 12104D-5
Forward 3 Long sequence 43 722 710 942
B
Reverse 4 Long sequence 49 720 718 617
} 3 (default) Actinomyces naeslundii 100% 692 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI standard nucleotide database 3 (default) Actinomyces naeslundii 100% 1,483 0

5 Forward 1 Long sequence 48 761 760 611

A
Reverse 2 Long sequence 50 754 759 2,067
} 3 (default) Akkermansia muciniphila 100% 725 0

Akkermansia muciniphila
ATCC BAA-835D-5
Forward 3 Long sequence 32 722 683 245
B
Reverse 4 Long sequence 43 701 703 285
} 3 (default) Akkermansia muciniphila 100% 689 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly blast into NCBI microbial portal 4 Akkermansia muciniphila 99.79% 1,434 0

6 Forward 1 Long sequence 47 770 760 558

A
Reverse 2 Long sequence 46 793 776 2,719
} 3 (default) Streptococcus agalactiae 100% 761 0

Streptococcus agalactiae
ATCC BAA-1138D-5
Forward 3 Long sequence 46 719 714 2,863
B
Reverse 4 Long sequence 48 725 729 5,812
} 3 (default) Streptococcus agalactiae 100% 689 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 4 Streptococcus agalactiae 99.79% 1,471 0
Each consensus sequence was aligned using the NCBI Short sequencing runs
Microbial Genomes BLAST tool that contains the genome The same set of DNA samples used for long sequencing
sequences of over 7,000 bacterial species. All BLAST runs was again used for another PCR or sequencing run,
queries yielded the expected species identification at very and electrophoresed using the short sequencing run mode
high (>99.5%) identity rate with the exception of sample no. on the SeqStudio instrument. The short sequencing run
4. Here the Actinomyces naeslundii sample was identified only takes about 30 min for the 4 sequencing reactions
as Actinomyces oris but with lower identity values and with and yields a total of over 2,000 nucleotides with high-
gaps in the alignment. These data result from an anomaly quality values. About 40% of the sequences covered both
in the sequence deposition in databases. At the time these forward- and reverse-direction strands and about 60%
experiments were performed, the genome for A. naeslundii of the sequence covered only one direction (Figure 8).
was not entered in the NCBI Microbial Genomes database The sample score values for the single-coverage portion
yet, but the 16S rRNA gene sequence was available in were usually greater than 40 and thus exhibited very high
the NCBI standard nucleotide database. When these confidence in base call accuracy. Table 4 shows that all
sequences were aligned using the NCBI standard samples were identified correctly above 99.5% identity
nucleotide database, the correct identification at a 100% rate using the consensus sequences for PCR segments
identity match was obtained. Therefore, great care should “A” or “B” alone or in combination: “A joined with B”.
be taken to choose the appropriate and valid database for This demonstrates that a full 16S gene sequence can
the 16S rRNA gene sequence queries as certain microbial be obtained for a given specimen with a short CE run of
species may differ by only a few nucleotides in their 30 min (compared to about 105 min with a long CE run).
16S genes, may be incorrectly listed, may not be included
at all, or may be listed under a different species name.

Figure 8. Overview of the contig assembly for short run and long run sequencing files for sample number 27 (Acinetobacter baumannii). The
long run (105 min) yielded almost full bidirectional coverage whereas the short run (30 min) yielded full 1x coverage and about 40% bidirectional coverage.
Table 4. Summary of short CE run sequence data metrics, alignment results, and identification of the microbial DNA test panel representing
8 species from 5 bacterial phyla.

Sequencing trace metrics MicrobeBridge software BLAST results

DNA input of known PCR Sequencing Sequencing SeqStudio Sample Signal Trim setting for contig No. of nt No. of
organism (sample) primer set primer trace No. run mode score CRL QV20+ RFU assembly Top ID Identity (%) aligned gaps

1 Forward 1 Short sequence 51 535 538 3,890

A
Reverse 2 Short sequence 50 521 531 1,696
} 3 (default) Bacteroides fragilis 99.87% 756 0

Bacteroides fragilis
ATCC 25285D-5
Forward 3 Short sequence 47 539 540 2,935
B
Reverse 4 Short sequence 50 532 534 1,394
} 3 (default) Bacteroides fragilis 99.56% 677 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 4 Bacteroides fragilis 99.72% 1,451 0

2 Forward 1 Short sequence 50 538 544 3,375

A
Reverse 2 Short sequence 48 539 539 2,558
} 3 (default) Fusobacterium nucleatum 99.73% 734 0

Fusobacterium nucleatum
ATCC 25586D-5
Forward 3 Short sequence 46 529 536 2,843
B
Reverse 4 Short sequence 50 528 540 2,783
} 3 (default) Fusobacterium nucleatum 100% 690 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 3 (default) Fusobacterium nucleatum 99.86% 1,442 0

3 Forward 1 Short sequence 50 530 541 2,535

A
Reverse 2 Short sequence 48 529 532 2,106
} 3 (default) Acinetobacter baumannii 100% 752 0

Acinetobacter baumannii
ATCC 17978D-5
Forward 3 Short sequence 45 532 533 2,139
B
Reverse 4 Short sequence 49 525 538 2,476
} 3 (default) Acinetobacter baumannii 100% 689 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 3 (default) Acinetobacter baumannii 100% 1,459 0

4 Forward 1 Short sequence 50 526 530 2,729

A
Reverse 2 Short sequence 49 542 543 1,632
} 3 (default) Actinomyces naeslundii 100% 773 0

Actinomyces naeslundii
ATCC 12104D-5
Forward 3 Short sequence 44 536 541 2,035
B
Reverse 4 Short sequence 50 517 536 1,948
} 3 (default) Actinomyces naeslundii 100% 692 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI standard 3 (default) Actinomyces naeslundii 100% 1,483 0

5 Forward 1 Short sequence 50 535 543 2,253

A
Reverse 2 Short sequence 47 522 531 1,561
} 3 (default) Akkermansia muciniphila 100% 725 0

Akkermansia muciniphila
ATCC BAA-835D-5
Forward 3 Short sequence 42 526 527 1,390
B
Reverse 4 Short sequence 48 525 544 1,322
} 3 (default) Akkermansia muciniphila 99.86% 691 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 3 (default) Akkermansia muciniphila 99.79% 1,434 0

6 Forward 1 Short sequence 50 539 536 4,107

A
Reverse 2 Short sequence 49 529 531 2,108
} 3 (default) Streptococcus agalactiae 99.74% 761 0

Streptococcus agalactiae
ATCC BAA-1138D-5
Forward 3 Short sequence 45 515 526 2,825
B
Reverse 4 Short sequence 49 528 540 3,346
} 3 (default) Streptococcus agalactiae 100% 689 0

Sequencing traces No. 1 + 2 + 3 + 4 for contig assembly BLAST into NCBI microbial portal 3 (default) Streptococcus agalactiae 99.86% 1,471 0
Conclusions
In this application note, we demonstrated the feasibility of
sequencing the 16S rRNA gene on the SeqStudio Genetic
Analyzer for the purpose of bacterial identification. The
16S Direct workflow that was presented here for its use
on the SeqStudio instrument could also be applicable on
other Applied Biosystems™ CE instruments such as the
3130 or 3500 series instruments with similar run modules.
The 16S Direct workflow includes a novel and streamlined
primer design that reduces the number of PCR amplicons
to 2 and the number of sequencing reactions to 4 to obtain
near full-length DNA sequence information for querying
genomic databases such as the NCBI Microbial Genomes
or CDC MicrobeNet. To that end, the BigDye Direct PCR
and sequencing reagents generate high-resolution and
high-quality sequence reads right from the beginning of the
primer. A short CE sequencing run of 30 min loaded with
4 sequencing reactions is sufficient to yield approximately
2,000 high-quality bases (i.e., 4 x ~500 bases) for
subsequent 16S gene contig assembly using the free
MicrobeBridge software. This fast and economical
workflow will benefit microbiology laboratories that use
DNA sequencing as their ultimate bacterial identification
and characterization tool. The affordable, compact, and
versatile SeqStudio Genetic Analyzer makes it easy to
introduce molecular genetics techniques in the general
microbiology laboratory.
Ordering information
Product Cat. No.
SeqStudio Genetic Analyzer A35644
MicrobeBridge software A29238
BigDye Direct Cycle Sequencing Kit 4458687
BigDye Terminator v1.1 & v.3.1 5X Sequencing Buffer 4336697
BigDye XTerminator Purification Kit 4376486
ProFlex 3 x 32-well PCR System 4484073
PrepMan Ultra Sample Preparation Reagent 4318930
E-Gel General Purpose Agarose Gels, 2% G501802
TE Buffer 12090015
Nuclease-Free Water AM9937

References Additional information

1. Woese CR et al. (1990) Towards a natural system of organisms: proposal for the
Applied Biosystems™ MicroSEQ™ Microbial
domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 87:4576-4579.
2. Clarridge JE 3rd (2004) Impact of 16S rRNA gene sequence analysis for identification Identification System
of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev Please note that the use of MicroSEQ ID products
17:840-862.
in combination with the SeqStudio Genetic Analyzer
3. Woo PC et al. (2008) Then and now: use of 16S rDNA gene sequencing for bacterial
identification and discovery of novel bacteria in clinical microbiology laboratories.
is for Research Use Only; not verified or supported
Clinical Microbiol Infect 14: 908-934. for pharmaceutical QC purposes. Find out more at
4. Petti CA et al. (2008) Interpretive criteria for identification of bacteria and fungi by DNA thermofisher.com/microseq.
target sequencing; Approved Guideline. Clinical and Laboratory Standards Institute.
CLSI Document MM18-A 28(12).
5. Weisburg WG et al. (1991) 16S ribosomal DNA amplification for phylogenetic study.
J Bacteriol 173: 697-703.

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thermofisher.com/seqstudio
For Research Use Only. Not for use in diagnostic procedures. © 2019, 2020 Thermo Fisher Scientific Inc. All rights
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