Presentation made during the Nanopore Day meeting in Paris that took place March 15th 2018 from École normale supérieure Biology Institute on RNA sequencing and data analysis using the MinION system from Oxford Nanopore Technologies.

1. RNA sequencing and data
analysis using the MinION system
Stéphane LE CROM @slecrom
Sorbonne Université
École normale supérieure Biology Institute
@NanoporeConf #NanoporeDayParis March 15th 2018

2. RNA-seq
small
amounts
25%
RNA-seq
poly A
40%
RNA-seq
ribosomal
depletion
3%
ChIP-
seq
4%
Home
made 27%
Tests 1%
IBENS genomic core facility
We help researchers working on high
throughput genomic projects from experimental
design to publication.
We do functional genomics in eukaryotes
• Whole transcriptome – RNA-seq
• Chromatin IP – ChIP-seq
We validate our protocols using a mouse
model of peripheral nervous system
development where we compare 2 conditions:
Krox20 (Egr2) KO that blocks myelination VS
Wild Type strains.
#NanoporeDayParis - March 15th 2018 2
Library preparation types

3. We automated MinION run primary analysis
We developed tools to automatically
launch data transfer and basecalling.
We benchmark for optimal threads
usage before using Albacore.
SSD storage is mandatory to quickly
basecall 1D2 runs (from 30 to 7 days).
#NanoporeDayParis - March 15th 2018 3
Acquisition
RAID 1 + UPS
Storage
85 TB
Processing
6x 16 cores - 196 GB
https://hub.docker.com/r/genomic
pariscentre/albacore/

4. We developed ToulligQC for RNA-seq evaluation
ToulligQC efficiently handles files to
quickly produce a run QC, is adapted to
RNA-seq and takes barcoding into
account.
Outputs:
• yield plot to check sequencing
homogeneity;
• transcript length histogram;
• barcode proportion plot;
• flowcell map to visualize spatial biases.
ToulligQC is compatible with Albacore 2 and
1D2 runs.
ToulligQC is available on github and as a
Docker image.
#NanoporeDayParis - March 15th 2018 4
https://github.com/GenomicParisCentre/toulligQC

5. We tested for barcoding influence on sequencing
When sequencing one sample by run,
barcoding is optional but should help
detecting biases or contaminations.
How run output behaves if we
systematically barcoded all our
samples?
Barcoding procedure does not have a
visible influence on the overall throughput
of sequencing.
#NanoporeDayParis - March 15th 2018 55
BC1-WT1 WT1
2D 1D
0
1
2
3
4
5
6
7
8
Feb 2017 Apr 2017 Apr 2017 May 2017
Millionofreadssequenced
without barcode with barcode

6. Barcodes could facilitate data analysis
We mapped samples on Ensembl 88
cDNA sequences using Eoulsan.
The myelin gene Mpz is the most
expressed transcript in barcoded samples
but not without barcode.
Myelin transcripts achieved similar number
of reads in both sample types.
Barcoding could help filtering out low
quality sequences
#NanoporeDayParis - March 15th 2018 66
Most expressed feature
Proportion of reads (%)
Barcoded
Mpz
Mpz
rRNA
rRNA
Actin BWithoutBarcode
Mpz
http://outils.genomique.biologie.ens.fr/eoulsan/

7. 0
1
2
3
4
5
6
7
8
08/2016 01/2017 03/2017 04/2017 05/2017
Millionofreadssequenced
By multiplexing we sequence all samples in one
MinION run
Our mouse model compares
2 conditions (KO vs WT) in
triplicates (100 ng total RNA).
We ran MinION flow cells (48h
runs) with this multiplexed design.
We achieved an average of
5.6 million reads with R9.4 flow
cells and 1D method.
The yield improvement in our
experiments reflects the quick
evolution of Nanopore
technologies.
#NanoporeDayParis - March 15th 2018 7
R9
2D 1D
R9.4

8. We compared MinION results to NextSeq short
reads
Minimap2 gives a more homogenous
coverage, longer alignments and ~2x
more read counts compare to BWA.
We get 6,551 differentially expressed
transcripts (adjusted p-value < 0.01) with
300,000 1D unique alignments by
sample.
86% of these transcripts are shared with
the one coming from Illumina.
#NanoporeDayParis - March 15th 2018 8
Log2FC
MinION
Log2FC
NextSeq

9. Detection of splicing events
Collaboration with GenoSplice to detect
new splicing events by comparing ONT
with Illumina reads.
We found Tropomyosine (Tpm3)
transcripts not seen using short reads.
#NanoporeDayParis - March 15th 2018 9

10. Concluding remarks and future directions
An automated procedure for data analysis;
Read number increasing along time, up to
10 million reads.
No visible influence of the barcoding procedure.
Differential analysis achievable with only
100,000 reads per sample.
On-going tests with direct RNA protocol (2 µg total
RNA = 900K reads in 24hrs - still running).
Tests to work with smaller amounts of starting
material.
#NanoporeDayParis - March 15th 2018 10

11. The IBENS genomics facility team
https://genomique.biologie.ens.fr
genomique@biologie.ens.fr Genomique_ENS
#NanoporeDayParis - March 15th 2018 11
Aurélien
Birer
Laurent
Jourdren
Fanny
Coulpier
Sophie
Lemoine
Ammara
Mohammad
Lionel
Ferrato
Cédric
Fund
Corinne
Blugeon
Bérengère
Laffay