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Variant Libraries
COMBINATORIAL | SOLD | SITE SATURATIONUNCOVER MORE WITH TWISTDRUG DISCOVERY.
ANTIBODY AND ENZYME ENGINEERING.
PROTEIN ENGINEERING AND STABILIZATION.

DIFFERENT APPLICATIONS, EACH WITH THE
SAME STARTING POINT. VARIANT LIBRARIES.
Twist’s high-quality libraries make it possible for you to explore a diverse range ofpotential variants and identify every hit, instead of just a few. Our years of molecularbiology expertise, and dedication to what we create, ensures that you uncover moreof what you want, and only what you want.
To create the libraries you want, we’ve used Twist’s proprietary silicon-based
synthesis platform, leveraging massively paralleled oligonucleotide synthesis toprint each variant base-by-base enabling precise synthesis of desired variants at
user-defined ratios without restriction sites and unwanted motifs that can impact
downstream processes. This has allowed us to develop diverse, high-quality libraries
that are just what you need to streamline your screening process, identify hits faster,and achieve higher success rates.
All of the NGS-verified quality control is done in-house, ensuring that both the input
and output are high-quality and clean, and that all desired variants are present.

TWIST BIOSCIENCEVARIANT LIBRARIESTECHNOLOGY TO DISCOVER MORE
Twist’s proprietary oligo synthesis technology offers a unique approach to library creation. The silicon-based synthesis
platform generates high-quality oligo pools, which are used to build fully-customized, user-defined variant libraries on asingle chip.
Our molecular biology experts use this novel technology to develop Combinatorial Variant and Site Saturation Variant
Libraries that outperform those developed with less-precise methods.
With Twist libraries, every variant is designed in-silico and screened prior to synthesis, eliminating unwanted sequencebias, premature stop codons, and undesirable motifs.

D E S IG N

SITE SATURATION VARIANT LIBRARYN G S V E RI F I C A T I ON

Wild Type
Mutagenesis
Site SaturationDA

MASSIVELY PARALLEL SYNTHESISof hundreds of thousands of oligos

SYNTHESIZED OLIGOS

C... W Y

COMBINATORIAL VARIANT LIBRARY
Wild Type
MutagenesisMultiple Sites
StretchMultiple Stretches
Length Variation“The Library we’ve asked for was very complex. Based on our analysis,
the library we received was excellent. What we’ve designed is what we’ve obtained.”
D R . S A N D R IN E M O U T E L
M A N AGER O F T EC HN O L O GY, INS T I T U T C URIE
Recombinant Antibody Platform and TAb-IP Platform

TWIST BIOSCIENCEVARIANT LIBRARIESLIBRARIES THAT YIELD VIABLE VARIANTS
Combinatorial Variant Libraries
Our Combinatorial Variant Libraries are designed to create gene variants that express proteins with better stability,
binding affinity, and enzymatic activity. Whether it be antibody humanization libraries or controlled complexity discovery
libraries, our variant cassettes can be seamlessly incorporated into single or multiple scaffolds to yield >1010 variants.
DEGENERATE(NNK/NNS)

TRIM/TRIMERCONTROLLEDTWIST COMBINATORIAL
VARIANT LIBRARIES

Eliminates sequence bias

No

No

Yes

Number of codons available

32

20

All 64

Prevents undesirable motifs

NoNo

Yes

Allows codon optimization

NoNo

YesAvoids stop codonsNoYesYesHigh-Diversity Libraries
Optimize your antibody and protein-screening experiments using more extensive high-diversity libraries that eliminate
sequence bias and undesirable motifs. Study what you want without wasting time on multiple iterations and screening
rounds—spend that time exploring a much larger sequence space that contains more hits in areas of interest. And you canrest assured that these libraries are absent of premature polypeptide sequence truncation, as Twist libraries do not containstop codons.
NORMALIZED DISTRIBUTION OF OBSERVED AMINO ACIDS100AC90

DE80FGFREQUENCY70HI

60KL50M
N40PQ

30RS20TV

100WY
EXP

OBS

1EXP

OBS

2EXP

OBS

3EXP

OBS

4EXP

OBS

5EXPOBS

6EXPOBS

7Example Application: CDR-H3 is largely responsible for the diversity among antibody structures and is critical for antigen binding. High-diversity
libraries remove bias often seen with other technologies such as NNK and Trimers. The diversity of this cassette enables a more comprehensive
interrogation of gene variant space.

TWIST BIOSCIENCEVARIANT LIBRARIES

User-Defined CDR Libraries
Our Precision Variant Library allows you to choose what unique CDR (complimentary defining regions) sequences you
want to be incorporated into the choice of framework(s). Each CDR can be codon-optimized to avoid the creation of
unwanted restriction sites. Machine learning has become an integral part of scientific research and has been used asa tool to analyze antibody libraries and identify unique CDR combinations that would yield, for example, higher affinityand specificity. Coupled with Twist’s silicon-based synthesis platform, explicit library combinations generated from theanalysis can be synthesized and seamlessly incorporated into a fully synthetic library to refine the exploration of thevariant space. Since every library is NGS QC’d, negative data can be used to identify mutations that do not yield improved
functions, and those can be removed in the next iteration of library design.
DESIGNITERATESCREEN

CDR1

1,000 unique CDR1 variants

CDR2

1,000 unique CDR2 variants

CDR3

10,000 unique CDR3 variants

Non-variant region

SOLD (Spread-Out Low Diversity) Libraries
Twist’s SOLD Libraries are a time- and money-saving tool for mapping protein sequences, allowing you to explore thecomplex relationship between a protein and its environment. It enables targeted exploration of the sequence space
encoded by mutating multiple positions simultaneously scattered across the parental sequence to generate a diverse
library of proteins.
These high-fidelity libraries are a fast and cost-effective alternative to NNK, trinucleotide mutagenesis (TRIM) technology,
error prone PCR, random mutagenesis and DNA Shuffling. Unlike the Combinatorial Variant Libraries (CVLs), SOLD
Libraries offer precise incorporation of diversity across the parental sequence without the restriction of variant domains.
In addition, SOLD Libraries allow you to explore multiple mutations simultaneously, unlike Site Variant Libraries (SVLs).

DFDDEIEEIE

SHL

SQRPW

VP

TWIST BIOSCIENCEVARIANT LIBRARIES

Site Saturation Variant Libraries
Twist’s Site Saturation Variant Libraries allow you to investigate the relationship between sequence and protein structureand function. These libraries are capable of generating 99% of desired variants and come in pooled or pooled-perposition options. They enable screening of 1–20 different amino acids (and up to 64 codons) at each position and acomprehensive investigation of sequence variants.
ERROR PRONE PCR

DEGENERATE(NNK/NNS)

TWIST SITE SATURATIONVARIANT LIBRARIES

Eliminates sequence bias

No

No

Yes

Number of codons available

Unknown32

All 64

Prevents undesirable motifs

No

No

Yes

Allows codon optimization

No

No

Yes

Avoids stop codons

No

Yes

Yes

Site Saturation Variant Libraries enable efficient sampling of a protein’s sequence space in screening assays. The figure
below is data from a Twist Site Saturation Variant Library with variants at 65 positions (19 variants per position). The barsrepresent a different amino acid position, with each color indicating the observed variant frequency. All variants arepresent in the expected ratios.
10090

CUMULATIVE % AA REPRESENTATION

80

70

60

50403020100ACDEF

GHIK

L

M

NP

QRS

T

VW

Y

TWIST BIOSCIENCEVARIANT LIBRARIESBUILT-IN QUALITYAt Twist, we use our molecular biology expertise to precisely and efficiently construct variant libraries. Our single-basecontrol approach allows us to deliver high-diversity libraries without motifs that could confound your screening process.
We deliver fully-customized libraries of unparalleled quality, with desired variants present at user-defined ratios.
Below you’ll see a CVL example representative of that quality. Variants in seven sequential amino acid positions weregenerated, and all have expected variants at the positions shown, with nearly all at the desired ratio:· At positions 1 and 6, the wild type amino acid was requested at 40% (position 1) and 30% (position 6). The remaining
18 amino acids were all requested to be as low as 3.3%.
· At position 3 to 5, all 19 amino acid residues were requested and observed at 5.3%.
OBSERVED AMINO ACID DISTRIBUTION
100%

A
C

90%

D
E

80%

F
G

70%

H
I

FREQUENCY

60%

K
L

50%

M
N

40%

P
Q

30%

R
S

20%

T
V

10%
0%

W
Y
EXP

OBS

1

EXP

OBS

2

EXP

OBS

3

EXP

OBS

4

EXP

OBS

5

EXP

OBS

6OBS

EXP

7

Twist Bioscience’s silicon-based platform enables the fabrication of customized libraries at the base-by-base level. Theability to precisely capture the variant diversity enables a comprehensive screen of the variant space while eliminating
the risk of missing key variants.

“Working with Twist...was a great experience...[Twist’s] support was amazing.
We have performed the first validation selections which looks promising...”
D R . A H U VA NIS SIM
P R O F E S S O R IN A N T IB O DY A ND T HER A P EU T IC EN GINEERIN G,
QUEEN M A RY UNI V ER SI T Y O F L O ND O N

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DOC-001176 Revision: 1.0