Taille:
INSTITUT DE BIOLOGIE DE L'ÉCOLE NORMALE SUPÉRIEURE

Services

We offer services in genomics and especially in high-throughput sequencing. We are specialized in functional genomics applications in all eukaryotes, particularly in Transcriptomics: RNA-seq (low input, long-read, single-cell) and Epigenomics/Epitranscriptomics: ChIP-seq, RNA m6A methylation. Our expertise also lies in long-read sequencing (Nanopore).

We also propose a sequencing service “ready-to-load” for users who make their own libraries.

Eukaryotic transcriptome (RNA-seq)

We propose a RNA-seq service with short-read Illumina sequencing (NextSeq 500) and long-read sequencing (Oxford Nanopore Technologies MinION).

General applications: RNA-seq data can serve to quantify RNA, measure differences in gene expression or isoforms, analyse splicing variants or polymorphisms, assemble de novo transcriptomes (if the genomic sequence is not available or with fragmentary annotation).

Samples: We work with many eukaryotic animal species, whether they are model organisms or not (human, rat, mouse, fish, frog, fly, butterfly, jellyfish, yeast, plants…). Samples are total RNA (we do not perform the step of RNA extraction), which can be obtained from cells (included sorted cells), tissues (included fixed), biopsies…

Proposed services:

  • Assistance with experimental design,
  • RNA quantification, RNA quality control,
  • Library construction (directional and non-directional) for short read Illumina sequencing, coupled with mRNA purification (polyA+) or rRNA depletion (capture of coding and non-coding RNAs) or a protocol for low input mRNA (polyA+),
  • Library construction (non-directional) for long read Nanopore sequencing, coupled with mRNA purification (polyA+), included for low input mRNA (polyA+),
  • Quality control of libraries, followed by sequencing in single-end or pair-end or long reads (depending on application),

  • Included automated bioinformatics analyses (Illumina): demultiplexing, quality control of the reads with report, adapter trimming, read mapping, quantification and differential expression for simple designs (pairwise comparisons),
  • Optional bioinformatics analyses (Nanopore):  basecalling, demultiplexing, quality control of sequences,
  • Deposition of results in public databases (GEO, NCBI) prior to publication,
  • Other analyses performed by a bioinformatician: usage of an annotated genome not accessible from the Ensembl database, complex experimental designs, non-standard parameters for the analyses, Nanopore analyses including isoform detection.

Single-cell eukaryotic transcriptome (scRNA-seq)

We propose a scRNA-seq service (10x Genomics) with short read Illumina sequencing (NextSeq 500) and are developing the long read sequencing (Oxford Nanopore Technologies) enabling to study isoforms at the single cell level.

General applications: scRNA-seq data can serve to explore the heterogeneity of cells within a tissue (including tumors), detect sub-populations or rare cell types.

Samples: We work with many eukaryotic animal species, whether they are model organisms or not (human, rat, mouse, fish, frog, fly, butterfly, jellyfish, yeast, plants…). Samples are either fresh cells dissociated or sorted (only within IBENS), or fixed cells (we are involved in the development of an approach using the ACME protocol).

Proposed services:

  • Assistance with experimental design,
  • Counseling for sample preparation,
  • Users within IBENS : usage of the 10x genomics Chromium by users,
  • Users external to IBENS : delivery of fixed samples (under development),
  • Library construction for short read Illumina sequencing, quality control of libraries, followed by pair-end  sequencing,
  • Library construction for long read Nanopore sequencing (under development),

  • Included automated bioinformatics analyses : demultiplexing, quality control of the reads with report,
  • Other analyses performed by a bioinformatician: read mapping of public or private genome (possibly including project-specific sequences like fluorescent marker), quantification with annotation, production of the CellRanger report (10x genomics).

Regulome (ChIP-seq, m6A)

We propose a service to construct the libraries for ChIP-seq with short read Illumina sequencing, and detection of modified nucleotides on RNA (m6A) with RNA direct sequencing (Nanopore).

General applications ChIP-seq: ChIP-seq data can serve to analyse protein/DNA interactions by detecting DNA regions enriched by chromatin immunoprecipitation. ChIP-seq enables to localize histone marks, and characterize transcription factor binding sites.

General applications m6A: Epitranscriptomics enables to identify and quantify modified nucleotides within RNA. The modified base m6A (N6-methyladenine) is the most common modification in eukaryotic mRNA.

Samples: We work with many eukaryotic animal species, whether they are model organisms or not (human, rat, mouse, fish, frog, fly, butterfly, jellyfish, yeast, plants…). ChIP-seq: samples  are precipitated chromatin. m6A:  Samples are total RNA (we do not perform the step of RNA extraction), which can be obtained from cells (included sorted cells), tissues (included fixed), biopsies…

Proposed services:

  • Assistance with experimental design,
  • Quantification of immunoprecipitated DNA or RNA, quality control of RNA,
  • Library construction for short read pair-end Illumina sequencing (ChIP-seq),
  • Library construction (non-directional) for long read direct RNA Nanopore sequencing (to detect m6A modifications), coupled with mRNA purification (polyA+), included for low input mRNA (polyA+),
  • Quality control of the libraries,

  • Included automated bioinformatics analyses (ChIP-seq) : demultiplexing, quality control of the reads with report, adapter trimming, read mapping,
  • Deposition of results in public databases (GEO, NCBI) prior to publication,
  • Other analyses performed by a bioinformatician: removal of low-quality reads,  detection of m6A modifications.

Service overview

I) Library preparation

Our genomcis core facility offers the following library preparation protocols:

  Short read library protocols
(Illumina)
Long read library protocols
(Oxford Nanopore Technologies)
RNA-seq PolyA mRNA purification
rRNA depletion (long non-coding RNAs)
PolyA mRNA purification
RNA-seq low input Low input amplification Low input amplification

scRNA-seq

10x Genomics 3’ Gene Expression 10x Genomics 3’ Gene Expression
scNauMi-seq (soon available)
Regulome

ChIP-seq

Direct RNA (m6A modification detection)

 

The following schema allow to choose the protocol against the biological question:

II) Sequencing

Our genomics core facility have sequencers from two supplier: Illumina for short reads sequencing and Oxford Nanopore Technologies for long reads sequencing.

We also propose a sequencing service “ready-to-load” for users who make their own libraries.

 

Short reads sequencing

Illumina sequencers allow to perform single and or paired-end sequencing. With paired-end sequencing, the two ends of the inserts between adapters are sequenced instead of just one in single end mode. In most the the cases, the length of the two ends are the sames (they are commonly noted like 2 × 75).

The GenomiqueENS core facility currenly use an Illumina NextSeq 2000 sequencer for short reads sequencing.

Illumina NextSeq 2000
Flowcell P2
(
~450 million reads per run)
Flowcell P3
(
~1.1 billion reads per run)
Kit Size (bp) Kit Size (bp)
N/A 50 cycles 1 × 50
2 × 25
100 cycles 1 × 100
2 × 50
100 cycles 1 × 100
2 × 50
200 cycles 2 × 100 200 cycles 2 × 100

 

 

Long reads seqencing

Oxford Nanopore Technologies sequencers allow to sequence DNA or RNA. The read lengths and the throughput vary against the sequencing mode:

Oxford Nanopore Technologies MinION
Kit Size (bp) Read count
DNA
(cDNA)
Until several thousands kb
(full length transcripts in RNA-seq)
6-8 million reads
Direct RNA ~1,000 bp mean size  ~1 million reads

 

III) Bioinformatics analyses

Our genomics core facility offers two service levels for bioinformatics analysis (the descriptions of the bioinformatics services are available in the application sections of this page):

  • Automated analysis included in the sequencing price, without the help of a bioinformatican: basecalling, demultiplexing, quality control of the reads, and more for some application type
  • An optional accompanied analysis, with the help of a bioinformatician

Bioinformatics analyses are only performed on data sequenced by our genomics core facility. To analyse your data, you can contact a bioinformatics core facility.

Proceedings of a collabirating project with our genomics core facility

Since 2006, we have created an “accompanied project” service as a scientific collaboration that handle all the experimental process and provide “read-to-publish results”. You benefit from our experience and our know-how from experimental design to the data analysis and an help to publication writing.

 

We are open to academic laboratories and companies. Most of our project are performed with teams outside the IBENS.