Induro™, a Novel Reverse Transcriptase for Nanopore Direct RNA
Sequencing with Significantly Improved RNA 5’ Coverage
Luo Sun, Yan Xu, Bradley W. Langhorst, Jian Sun, Nicole M. Nichols, Eileen T. Dimalanta, and Theodore B. Davis
New England Biolabs, Inc.

INTRODUCTIONRESULTS

Transcriptome analysis based on cDNA sequencing of direct or PCR amplified cDNA has limitations,
including generation of cDNA chimeras during library preparation, artifacts associated with templateswitching, DNA contamination and size bias during PCR amplification. Recent advances from OxfordNanopore Technologies (ONT) have enabled direct RNA sequencing without PCR. This method not onlyinterrogates full-length RNA molecules and generates higher resolution data on complex isoforms, but also
allows for the detection of RNA modifications. This opens up new opportunities for studying RNA stability andregulation. To perform direct RNA sequencing, one approach is to first convert RNA into an RNA/cDNA hybrid
molecule using a reverse transcriptase (RT). The RNA/cDNA hybrid molecule reduces RNA secondarystructure formation, leading to higher sequencing throughput and longer sequencing reads without sacrificing
RNA modification information. To accomplish this, an ideal reverse transcriptase would be highly processive
and generate high cDNA yields.
Here we introduce Induro, a novel reverse transcriptase that delivers superior performance in the cDNA
synthesis reaction. Using this novel RT, we generated a RNA/cDNA hybrid library starting with mouse brain
polyA-enriched RNA. After ligation with ONT sequencing adapter following the SQK-RNA002 protocol, thislibrary was sequenced on the GridION. When compared with other commonly used RTs, Induro produced
longer average read lengths and better 5’ end coverage of RNA transcripts. Similar results were also
obtained by performing direct sequencing on a cDNA library generated using this RT enzyme. In summary,Induro is a significant improvement compared to other available RT enzymes. It can produce longer cDNA
molecules, and exhibits higher cDNA yields, better full-length coverage, leading to more accuratetranscriptome analysis.

5’3’AAAAAA

Preparation timemRNA

SSIII
Total: 2 hrs
Hands on: 30min

RTA adapter ligation
5’InduroTotal: 1.2 hrHands on: 30min3’ TTTTTT
RTcDNARMXligation

Beads
clean up

• 500 ng input mRNA
• Ligate RTA adapter (SQK-RNA002 protocol) usingNEBNext ® Quick Ligation Module (E6056)• RT reaction: SuperScriptTM III (SSIII, 50 min at
50oC), Induro (15 min 60oC, 10 min 70oC)• Ligate RMX adapter using NEBNext QuickLigation Module (E6056)

3’5’Beads
clean up

RTreaction

RTA ligation

5’
RMX adapter ligation
3’

Oxford Nanopore (ONT) multiplex RNA Library Prep

3’

RTA barcodeligation

PoolBeads
clean up5’Final Library

RMXligation• 500 ng input mRNA
• Ligate RTA barcode adapter (SQK-RNA002 protocol)using NEBNext Quick Ligation Module (E6056)• RT reaction: SSIII (50 min at 50oC), Induro (15 min60oC, 10 min 70oC)• Samples were pooled together for multiplexing• Ligate RMX adapter using NEBNext Quick Ligation
Module (E6056)

B

Data Throughput- Unaligned reads

1,542

1.1%

- Unaligned reads 2,093- Aligned reads

145,229

98.9%- Aligned reads--- Single-alignreads

144,817

98.7%--- Gapped-alignreads

35--- Chimeric reads

Induro99.5% reads aligned
Read Stats

314,671

Total reads

506,826

0.7%- Unaligned reads

2,710

0.5%

312,578

99.3%- Aligned reads

504,116

99.5%--- Single-alignreads

311,605

99.0%--- Single-alignreads

502,473

99.1%

0.02%

--- Gapped-align
reads

107

0.03%

--- Gapped-align
reads

298

0.06%

377

0.26%

--- Chimeric reads 866

0.28%--- Chimeric reads

1,345

0.27%

----- Trans-chimeric
377
reads

0.26%

----- Trans-chimeric
863
reads

0.27%

----- Trans-chimeric
1,340
reads

0.26%

----- Self-chimericreads

0.00%----- Self-chimeric
3reads0.00%----- Self-chimeric
reads

0.00%

05

Base Stats (of aligned reads)

Base Stats (of aligned reads)

Base Stats (of aligned reads)

Total bases

155,340,385

Total bases

Total bases

782,448,115- Unaligned bases

10,349,985

6.7%- Unaligned bases 19,964,900

5.1%- Unaligned bases

35,776,076

4.6%- Aligned bases

144,990,400

93.3%- Aligned bases

374,361,179

94.9%- Aligned bases

746,672,039

95.4%--- Single-alignedbases

144,775,569

93.2%--- Single-aligned
bases

373,806,638

94.8%--- Single-aligned
bases

745,665,454

95.3%--- Other-aligned
bases

14,162

0.01%--- Other-aligned
bases

48,854

0.01%--- Other-aligned
bases

159,1300.02%

394,326,079

Mappedno_RT

AFull-length

GeneTranscript

Reads Sampled

Read Sampled

Read Sampled

Read Sampled

Read Sampled

Read Sampled

42.6%

SSIII
62.5%
58.4%

Induro
70.7%
65.6%

no_RT

SSIII

Alignment stats

Alignment stats

Best alignments
sampledBase stats
Bases analyzed- Correctly alignedbases- Total error bases--- Mismatchedbases

Figure 4. Alignment Analysis
All reads from 3 multiplex runs
were merged for no_RT, SSIII
and Induro libraries. Merged
data was mapped to themouse genome mm10 using
minimap2. Mapping quality
was assessed using Alignqc.
Result shows very similaralignment statistics for all 3samples. Induro and SSIII
libraries have a slightly higher
percentage of reads and basesaligning to the genome.
Chimeric read rates weresimilar for all 3 libraries.

Figure5.
Annotation
andrarefaction analysis. Mapped data
was analyzed using Alignqc for thenumber of detected genes andtranscripts for no_RT (A), SSIII (B),and Induro (C) libraries. The
percentage of full-length genes
detected is 70.7% from Induro vs
62.5% from SSIII and 45.0% fromno_RT libraries.
Similarly, thepercentage of full-length transcripts
detected was higher for Induro
libraries (65.5%) compared to SSIII(58.4%) or no_RT (42.6%) libraries.
This data demonstrates that higher
full-length genes and transcripts
were obtained with Induro.

45.0%

Beads
clean upRESULTS400

Total reads

Rarefraction stats
FeatureCriteriaCount
Genefull-length 9,519
Gene
any match 13,470
Transcript full-length 10,966
Transcript any match 16,733

Figure 1. Direct RNA sequencing libraries were prepared following the Oxford Nanopore SQK-RNA002 protocol. 500ng mousebrain polyA (TaKaRa, cat # 636207) was used as input to make RNA/cDNA libraries with either Induro or SSIII. RNA libraries
without cDNA synthesis (no_RT) were also generated. All three methods (no cDNA synthesis, or cDNA synthesis with Induro orSSIII) were used to make single-plex and multiplex libraries. Final Library yields were measured using Invitrogen™ Qubit™
dsDNA HS Assay Kit.

A

146,771

C5’

RT
reaction

Total reads

Rarefraction stats
FeatureCriteriaCount
Genefull-length 7,530
Gene
any match 12,046
Transcript
full-length 8,394
Transcript
any match 14,364

Oxford Nanopore (ONT) RNA Library prep
5’

Read Stats

B

3’

AAAAAA

Read Stats

Rarefraction stats
FeatureCriteriaCount
Genefull-length 4,610
Gene
any match 10,253
Transcript
full-length 4,951
Transcript
any match 11,611

METHODS

SSIII
99.3% reads aligned

no_RT
98.9% reads aligned

Best alignments
sampledBase stats
Bases analyzed- Correctly alignedbases- Total error bases--- Mismatched
bases

901
1,002,927
897,968

89.5%

104,959

10.465%26,2822.621%--- Deletion bases

49,2674.912%----- Complete
deletion bases

28,937----- Homopolymer
deletion bases

InduroAlignment stats
Best alignments
sampledBase stats
Bases analyzed- Correctly aligned
bases- Total error bases--- Mismatched
bases

801
1,050,043
945,09390.0%

104,950

9.995%

27,9132.658%--- Deletion bases

50,4774.807%

2.885%----- Complete
deletion bases

29,803

20,3302.027%----- Homopolymer
deletion bases--- Insertion bases

29,4102.932%----- Complete
insertion bases

18,786----- Homopolymer
insertion bases

10,624701
1,113,365
1,003,544

90.1%

109,8219.864%

29,2142.624%--- Deletion bases

52,6274.727%

2.838%----- Complete
deletion bases

30,6402.752%

20,674

1.969%----- Homopolymer
deletion bases

21,9871.975%--- Insertion bases

26,5602.529%--- Insertion bases

27,9802.513%

1.873%----- Complete
insertion bases

16,025

1.526%----- Complete
insertion bases

16,7451.504%

1.059%----- Homopolymer
insertion bases

10,5351.003%----- Homopolymer
insertion bases

11,2351.009%

Figure 6. Error statistics. Arandom
down-sampledsubset of sequencing datafrom no_RT, SSIII and Induro
were mapped to the mouse
genome mm10. Mapping
data was analyzed for errors
including mismatch, deletion,
insertion and homopolymers
using Alignqc. Similar error
rates
wereseenwithdifferent libraries.

# of Reads >4k70000

354350264250196200

175
134

150

108

40000

no_RT
SSIII
Induro30000

Induro

39

10000
3855

02

no_RT

C

no_RT

3

SSIII

SSIII

no_RT

Coverage

01

InduroTM

SSIII

InduroRT4
InduroDReads Length
2500

ELongest 5 Reads

SSIII

Length Distribution

25000

no_RT

2088

2068
1949

2000

20572
20000
17686

1347

1297

15623

bases

1474

bases

1682

1633

15000

1306

1000

12070

11136

12064

040

60

80

100

Gene Body Percentile (5’-3’)

9211

10000 8890

200

1020

30

40

50

60

70

80

90

100

Gene Body Percentile (5’-3’)

7104
5000

500

Table 1.

Direct cDNA Sequencing Using Different RT Enzymes001

no_RT2SSIII3

RT4no_RT

1mean

SSIII

2RT4no_RT

31SSIII

RT42N503no_RT

1SSIII2mean
N50

1. no_RT 2. SSIII 3. Induro

RT43no_RT

SSIII

RT431351224no_RT

1SSIII

2

RT43
no_RT

SSIII

Induro1. no_RT 2. SSIII 3. Induro

Figure 2. 3 sequencing runs were performed on pooled barcoded samples containing the direct RNA library (no_RT), theRNA/cDNA libraries made with either SSIII or Induro in the RT step. (A) Data throughput for each of the sequencing runs. TheInduro libraries have significantly and consistently higher base-called data (orange bar). Qubit measurements of double
stranded RNA/cDNA yield with Induro is higher than with SSIII, the ONT recommended RT (data not shown). (B) All reads
from 3 multiplexed runs and two single-plex runs/sample were merged together for data analysis. The merged data wasmapped to mouse mm10 genome using minimap2 . Reads (358Mb for no_RT, 3.6Gb for SSIII, 3.2Gb for Induro) were filteredfor reads with length >4kb. The number of reads >4kb long is 45% higher with the Induro library (orange bar, 2.6% of totalreads) than from SSIII (gray bar, 1.3% of total reads). (C) Data analysis for mean and median read lengths shows that theInduro libraries have significantly longer mean length and N50 values. The no_RT libraries have the lowest mean length andN50 values. (D) Analysis of top 5 longest reads demonstrates that Induro libraries have the longest reads up to 20kb,
significantly longer than reads from SSIII and no_RT libraries. (E) Boxplot of read length distribution shows Induro libraries
have longer median read lengths. Also observed is the significantly longer tail for Induro libraries, indicating more long reads.

A12

SSIII8no_RT

6

Mean read length (bp)Median read length (bp)
Number of reads
Read length N50 (bp)Induro SSIII
2562 2118.7
1896
1665
175400 97638
3658
2855

SSIV
Maxima TGIRT
2142
1893
2077
1682
1441
1611
148859 343925 144746
2926
2681
2859

Marathon
1971
1573
1708602726

ProtoScript II
1995
1561
1492402703

Table 1. Impact of RT enzymes on the length of cDNA reads following recommended conditions was studied using direct
cDNA sequencing on the GridION. In this experiment, after first strand cDNA was synthesized in the RT reaction, 2nd strand cDNA
was synthesized using NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module. Then the cDNA products were
purified and ligated to a barcoded adapter following the protocol of Nanopore EXP-NBD104 kit. cDNA library concentrations weremeasured on a Qubit after the barcoding step and then equal amounts of DNA were pooled and ligated with a sequencing adapter.
Finally, the resulting DNA library was cleaned up with beads and run on a GridION with a R9.4.1 flow cell. The data wasdemultiplexed and analyzed. Results are consistent with direct RNA sequencing data in that reads from the Induro library has thelongest mean, median and N50 values.

CONCLUSIONS

Mean Quality Scores

B

Reads Quality

10Quality Score

bases

Figure 7. Gene bodycoverage percentile
Gene Body Coverage (A)and a heat map (B) weregenerated using RSeQC. In
the heat map, the aqua color
indicates low coverage andpink indicates high coverage.
In both plots, it clearly showsthat Induro libraries havesignificantly
better
5’coverage than libraries madeusing SSIII or made without
cDNA synthesis (no_RT).

20000

5250

Better 5’ coverage for Induro

B

40513

80

100

Better 5’ coverage for Induro

A

50000

# of reads

Mbp

300

1500

58618

60000

Induro4• Induro, a novel reverse transcriptase shows superior performance for generating libraries for direct RNA
and direct cDNA Oxford Nanopore sequencing.
• Induro is very processive, requiring a short incubation time to generate full length cDNA.
• Longer read lengths, higher percentage of full-length genes and transcripts, improved 5’ coverage can beachieved with Induro compared to SSIII without increasing error rates or decreasing mapping rates.

20no_RT

1SSIII

2RT43

no_RT

1

mean

SSIII

2

RT43

no_RT

1

SSIII

2

median

1. no_RT 2. SSIII 3. Induromean
median

RT430250050007500

10000 12500 15000 17500

Position (bp)

Figure 3. Read Quality (A) Read quality is similar between the 3 different libraries, and varied more by sequencing run than bylibrary type. (B) Quality score as a function of base position is shown. Induro libraries maintained a higher mean quality scoreat lengths > 1kb compared to other samples.

ACKNOWLEDGEMENTS• We thank Dr. Maggie Heider for proof reading and Dr. Guoping Ren for the discussion and assistance indeveloping Induro.