Quality control by full-length deep sequencing

Introduction

Final PlasmoGEM vectors are screened by a combination of PCR and NotI restriction digest and quality controlled by full-length sequencing. QC uses Illumina technology on pooled batches of many vectors, batches of many vectors, which means only target specific sequence elements can be verified.

How we decide on QC pass or failure

Large deletions, as well as SNPs and indels in coding sequences are the most serious types of quality issues, because they may lead to unintended mutations in tagged or neighboring genes. This may lead to an incorrect association of phenotypes and genes. If we cannot provide a sequence perfect vector, we tolerate SNPs and small indels but only in non-coding regions. These are shown in the database.

QC pass

If a vector has passed QC, the sequence of its homology regions has been verified. Small indels or single base changes in non-coding genomic sequence in the homology arms and such vectors still pass QC. The majority of these mutations are single base insertions or deletions in long homopolymeric tracts of A/T nucleotides. Many of these will have originated in E. coli but others may pre-exist in the parasite clone from which the gDNA library was created. Small variations in non-coding regions of low complexity are difficult to avoid entirely and the vast majority will neither affect vector function, nor influence downstream phenotypic analysis of resulting transgenic lines. These constructs pass QC so they can be used where no sequence perfect vector is available. Please note that the sequence of the selection cassette in the final vectors is not verified, but we do not anticipate this to be a problem.

QC failure

Detection of any one of the below feature(s):

• Defective barcode module: The barcode, or one of the primer annealing sites flanking it, have a sequence discrepancy that was introduced during primer synthesis.
• Large deletion: The vector carries a deletion in one of its homology arms. Such deletions could reduce the chance of a vector integrating into the genome by forcing recombination to happen inside of the deletion, or deletions might be introduced into the genome in addition to your intended mutation and lead you to misinterpret your phenotype.
• Other fail reasons: The vector has a potential single base exchange or small indel that affects the protein-coding sequence or predicted splice site of any gene within the genomic locus.