Download NextGen DNA Sequencing for Cell Line Characterization and

Identification of human S/MAR elements to improve gene
expression and regulation
University of Lausanne
NextGen DNA Sequencing for Cell Line
Characterization and Potential to
Assess Clonality
!
Nic Mermod!
WCBP 2015, Washington DC, January 2015
Clonal variability of expression
Improved expression with MAR elements:
+
Transgene
E/P
MAR
Vector elements
GFP
GFP expression + MAR
GFP expression, no MAR
Doubling&,me&(h)&
one transfection
two transfections
50#
40#
30#
20#
0#
10#
20#
30#
40#
50#
60#
70#
80#
Clone&produc,vity&(picogram&/&cell&/&day&of&IgG)&
Integration-linked effects (e.g. transgene copy number)
Position effects (e.g. chromatin-linked silencing)
Clone / protein effects (e.g. clonal fitness)
Possible transgene copy mutations
Can we document integration loci, transgene integrity and monoclonality using NGS ?
Girod et al Nature Meth (2007); Galbete et al Molec Biosyst (2009); Grandjean et al NAR (2011); Arope et al PLoS One (2013); Ley et al PLoS One (2013); Majocchi et al NAR (2014); Le Fourn et al Metab Eng (2014)
Rationale for CHO-M genome sequencing
1.  For transgene sequence characterization
2.  To identify transgene integration loci and mechanism
3.  To possibly document the monoclonality of cell populations ?
Nic Mermod, WCBP – CASSS 2015
CHO Genome Sequencing and assembly
!  390 Gb raw CHO-M genome sequence (Illumina and PacBio, N =27kb)
!  Genomes of 8 therapeutic-producing clones sequenced (10X)
50
Gene anotation and transcriptome analysis
mRNA sequence and level for 16’289 CHO-M genes
5’748 genes essentially silent in CHO-M cells
98% of CDS common to CHO-M and K1
Can we identify the genomic integration sites of transgenes ?
Nic Mermod, WCBP – CASSS 2015
Our Whole-Genome Sequencing approach
Mixed plasmid vectors in various orientations and orders
…
R1
(+/- strand)
R1
(+ strand)
3’
5’
R2
(+ strand)
R1
(- strand)
R1
(+/- strand)
R1
(+/- strand)
R2
(+/- strand)
R2
(- strand)
R2
(+/- strand)
R1
(- strand)
5’
3’
R2
(+ strand)
Genomic (PE) short fragments
(200-600 bp)
…
Possibly deleted portion of chromosome
R2
(+/- strand)
Portion of CHO chromosome scaffold
with inserted transgenes
…
Align sequences at left and right junctions
Transgene-genome insert representation
Genome
…
Vector
Vector
Genome
…
Junctions PCR amplification and sequence validation
Nic Mermod, WCBP – CASSS 2015
Discovery of genomic integration sites
Identifying relevant NGS reads:
R1
about 360 bp (paired end)
or 3000 bp (mate-pair)
11021
R1 Read1 (forward):
about 100 bp
of vector DNA
R2 Read2 (reverse):
about 100 bp
of genomic DNA
LC vector
R2
534 9580 42
0
0
0
24
CHO genome
841
(no R2 match)
Mapping reads on CHO-M genome scaffolds:
2 integration loci by FISH / 2 found in silico & validated experimentally
BS-03, 20 chromosomes
Clone
BS-03
CHO-M caryotype, 20 chromosomes
Clone
BS-01
BS-01, 19 chromosomes
5-6 integration loci by FISH / 6 found in silico & validated experimentally
Nic Mermod, WCBP – CASSS 2015
Current clone reporting software (V2.1)
Software for automated
clone analysis and
reporting
Nic Mermod, WCBP – CASSS 2015
A clone without coding sequence mutation
Known CMV enhancer change introduced by biotechnologist, but unknown to the bioinformatics analyst
Transgene coding sequence
coverage
Expected change in CMV enhancer, no other coding sequence change detected
Example of a good candidate clone to be pursued with
Nic Mermod, WCBP – CASSS 2015
A need for CHO clones sequencing ?
NGS can assess:
!  Transgene integrity at fusions with genome
!  Transgene copy number and correct sequence
!  Number and locations of genomic integration loci
!  Possible adverse effects from transgene integration
Providing early genomic validation during clone selection
Does NGS tell us something about clonality ?
Nic Mermod, WCBP – CASSS 2015
Propagation and detection of sequence variations
Cell line founder cell
= single-copy gene to whole genome
1
= Single Nucleotide Variants to SNV sets
Two-cell stage
2
3
4
Etc., etc.
Occurs in 45% of sequences
Occurs in 25% of sequences
Reliably detected with 20-fold coverage (cell line ‘barcode’)
Occurs in 12.5% of sequences
Possibly observed
Occur in 6.3% of sequences
Likely missed
Nic Mermod, WCBP – CASSS 2015
Cell line original genome determination
Original single CHO cell:
Parental CHO cell line:
Multiple cell genome NGS
Sequencing depth (i.e. fold coverage, on average)
Reference cell line genome sequence
Nic Mermod, WCBP – CASSS 2015
SNVs in CHO single copy gene
Original single CHO cell:
Parental CHO cell line:
Occurs in 45% of sequences
Occurs in 25% of sequences
Occurs in 12.5% of sequences
Occur in 6.3% of sequences
10-fold average coverage, single-copy gene, CHO-K1 cells
Set of 10 key DNA repair genes analyzed for SNVs by NGS
(maintain genomic sequence integrity)
Example of SNV with 25% frequency (3/12)
on single-copy gene
Exons as orange bars
Blue circles: at least 2 reads have 1 different nucleotide (single nucleotide variants) – minor allele
Red circles: at least 3 reads have 1 different nucleotide (single nucleotide variants) – minor allele
Green circles: at least 4 reads have 1 different nucleotide + near allelic proportion (>50%) - possible SNP
Example of SNV with 45% frequency (5/11)
on single-copy gene
Nic Mermod, WCBP – CASSS 2015
Transition of SNVs into fixed SNPs in clones
Original CHO cell:
= single-copy CHO gene
Parental CHO cell line:
SNV occurs in 45% of sequences
Transfection, cloning
SNVs occur in 25% of sequences
Clone A:
Clone B:
SNP detected in up to 100% of sequences
Detected in nearly 0% of sequences
Detected in nearly 0% of sequences
SNP detected in up to 100% of sequences
Detected in up to 50% of sequences (or possibly nearly 100% if 2nd cloning step)
Detected in up to 50% of sequences
Nic Mermod, WCBP – CASSS 2015
Transition of SNV into fixed SNP in BS01 clone
Original CHO cell:
= single-copy CHO gene
Parental CHO cell line:
Transfection, cloning
Clone A:
SNP detected in nearly 100% of sequences
SNV occurs in 50% of sequences
SNVs occur in 25% of sequences
Clone B:
SNP detected in nearly 100% of sequences
Parental (11/25 with SNV)
Derived
(11/11 with SNP)Derived clone (9/9 with SNP)
Parental
(0/27 clone
with SNV)
Nic Mermod, WCBP – CASSS 2015
The Issue of CHO cell clone ‘clonality’
Clone A:
Clone B:
Detected in 100% of sequences
Detected in 0% of sequences
Detected in 0% of sequences
50:50 mix of Clone A and B:
Detected in 50% of sequences
Detected in 50% of sequences
Detected in 100% of sequences
Mixed SNPs indicative of mixed
clones, for single-copy genes
None detected in 100% of sequences
What if the two clones don’t grow at the same speed ?
40 days from last cloning step, and with a 20-fold coverage:
40% difference in division time (19 vs. 27h): 99.997% of Clone A (fast grower), oligoclonality undetectable
10% difference in division time (19 vs. 20.9h, average difference): 96:4 mix of Clone A and B, oligoclonality missed
5% difference in division time (19 vs. 20h, unusually small, 25% of cases or less): 83:17 mix of Clone A and B
Even in the later rare case, 5% chance of missing oligoclonality with 20-fold coverage
(nB=0, p=0.05)
Nic Mermod, WCBP – CASSS 2015
Detection of non-clonal populations ?
Clone A:
Clone B:
Integration loci A in 100% of genomes
Average growth difference
Integration loci B in 100% of genomes
Mix of Clone A and B:
Loci A in 96% of sequences
First single cell isolation step (LD1)
Loci B in 4% of sequences
(LD2)
An affordable way out ?
Obtain 50 subclones, sequence ONE with a 20-fold coverage and determine the transgene integration loci
Experimentally validate the junction sequences and genomic integration loci of ONE subclone
PCR Amplify to check that the same integration loci occur in ALL 50 subclones
If YES, about 98% likelihood of monoclonality of LD1 population
Can assess that the LD1 process yields true clones
Nic Mermod, WCBP – CASSS 2015
What did we learn about population clonality ?
What is a clone ?
Is a clone a cell population with identical genomes ? - No
Are clones cell populations with single transgene integration site ? - No
Are clones cell populations arising from a single isolated cell after transfection ? - Yes
Do clones originating from a single transfected cell have identical transgene sequences ? - No
Can we assess “monoclonal-like properties” of populations by analyzing SNV fixation into SNPs ? - Yes
Nic Mermod, WCBP – CASSS 2015
Detection of non-clonal populations ?
What is a clone, and is it important to ascertain monoclonality ?
Monoclonal populations are collections of dissimilar cells submitted to darwinian evolution (clades)
A cellular population truly originating from a single cell does not insure genetic homogeneity
Genetic homogeneity of the genome and transgenes may be the most relevant PTC
NGS can check integrity of the cellular genes that prevent mutation occurrence
NGS ideally suited to assess transgene sequence without much bias
Might demonstrating sequence homogeneity and stability be more relevant than clonality ?
Should NGS be used routinely to assess cell identity and low mutation rate ?
Nic Mermod, WCBP – CASSS 2015
On commercial offers and disclosure time
! Platform for efficient therapeutic-producing clones
! CHO-M cell line genome and transcriptome NGS-analyzed
! Early stage clone validation for transgene sequence integrity
! Routine characterization of clonal genome sequence identity
! Perspectives to identify adventitious agents and assess their absence
Nic Mermod, WCBP – CASSS 2015
Institute of Biotechnology
Niko Niederländer
Yves Dusserre
Stéphanie Renaud
Jacqueline Masternak
Stefania Puttini
Ruthger van Zwieten
Kaja Kostyrko
Xuan Droz
Laurie Girard
Deborah Ley
Simone Edelmann
Thomas Junier
Elena Aritonovska
Solenne Bire
Pavithra Iyer
Nic Mermod
Acknowledgements
Collaborations
Selexis: I. Fisch, P.-A. Girod,
A. Regamey, V. Le Fourn
Clone analysis and engineering
Bioinformatics
SIB: I. Xenarios, T. Junier, N. Guex
Ch. Iseli, S. Neuenschwander
Emanuel Schmidt
Project funding bodies
KTI/CTI Swiss Government agency
Selexis SA
UNIL
Copyright Alain Herzog