TITLE: Validating  Diversity in DNA Libraries through NGS 
AUTHORS: Steffi Davison  

[DESCRIPTION]
DNA libraries are important resources to derive targets to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for a DNA library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection.

Quantitative evaluation of a DNA library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. 

Complexity was usually inferred by the transformation efficiency and tested by sequencing of a few random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. 

Next-generation sequencing (NGS) has opened new ways to tackle the DNA library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of DNA repertoires and to overcome current limitations. Even with the recent advances in NGS, it remains difficult to directly measure the representation of variant libraries, as the number of reads is insufficient to cover the size of a large library. As an example, a 1 kbp combinatorial DNA library with a billion variants has equivalent base pair content to that of 300 human genomes. Thus, brute force measurements of individual library members is impractical even with field-leading sequencing capabilities; i.e. >300 million reads at ~150 base lengths.


To obtain a more reliable DNA library complexity estimate, here we show a NGS approach to sequence DNA libraries on Illumina platform, coupled to with a bioinformatic analysis and software that allows to reliably estimate the complexity, taking in consideration the sequencing error


 


 


[STEPS]
SECTION: Complexity inferred by NGS of the library (library sequencing preparation)
2. The sample of the library, with length 0.5 µL, was prepared for “shotgun” sequencing on a high-throughput Illumina MiSeq sequencer (LBL)



 

SECTION: Complexity inferred by NGS of the library (library sequencing preparation)
3. For each sample, 3.75 µL  of library DNA was prepared in a 50 µL reaction with 0.5 µL of Illumina Nextera and 25 µL TD buffer at 55 ̊C for 5 minutes.

SECTION: Complexity inferred by NGS of the library (library sequencing preparation)
4. This reaction was then purified using a 0.6 x SPRI magnetic bead-based purification.


 

SECTION: Complexity inferred by NGS of the library (library sequencing preparation)
5. The resulting material appears on a gel as a smear of DNA with varying lengths between ~100–717bp. The material was also quantified on Qubit and Bioanalyzer.


 

SECTION: Complexity inferred by NGS of the library (library sequencing preparation)
6. The prepared DNA was used as template in a 50 µL index PCR with NEB Next mastermix and Illumina indexing primers; and re-purified using magnetic bead-based purification.

SECTION: Complexity inferred by NGS of the library (library sequencing preparation)
7. In final preparation, each sample was diluted to , before running on an Illumina MiSeq sequencer using reagents from an Illumina MiSeq Reagent KitV2, to produce 150 base paired-end reads.

SECTION: Sequence alignment to reference genome
8. Read mapping can be performed by two methods: (1) an implementation of Smith-Waterman alignment and (2) Bowtie2, an aligner based on the Burrows-Wheeler transform.

 


 

1. Library complexity inferred by the (1) transformation efficiency (Transformation) and/or (2) picking subset of clones/mutations for Sanger sequencing (Sample set) (example seen in Jha et al. 2019) or (3) sequencing the entire library (NGS library)


 

SECTION: Sequence alignment to reference genome
9. Sequence alignment with the reference sequence of the library used for this study appears in ICE and DIVA platforms. Namely, reference genome CJ019 (sequenced by Bill Alexander ANL 2022 using Nanopore).

  

SECTION: Sanger Sequencing
2. A small set of random clones were cultured, DNA extracted and sequence confirmed by Sanger sequencing
SECTION: Complexity inferred by the transformation efficiency and/or Sanger sequencing of a sample set
2. Count colonies to infer transformation efficiency