High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides.

Selective retrieval of sequence-verified oligonucleotides (oligos) from next-generation sequencing (NGS) flow cells, termed megacloning, promises accurate and reliable gene synthesis. However, gene assembly requires a complete collection of overlapping sense and nonsense oligos, and megacloning does not typically guarantee the complete production of sequence-verified oligos. Therefore, missing oligos must be provided via repetitive rounds of megacloning, which introduces a bottleneck for scaled-up efforts at gene assembly. Here, we introduce the concept of high-depth tiled oligo design to successfully utilize megacloned oligos for gene synthesis. Using acquired oligos from a single round of the megacloning process, we assembled 72 of 81 target Cas9-coding gene variants. We further validated 62 of these cas9 constructs, and deposited the plasmids to Addgene for subsequent functional characterization by the scientific community. This study demonstrates the utility of using sequence-verified oligos for DNA assembly and provides a practical and reliable optimized method for high-throughput gene synthesis.

Genomic Profile of Chronic Lymphocytic Leukemia in Korea Identified by Targeted Sequencing.

Chronic lymphocytic leukemia (CLL) is extremely rare in Asian countries and there has been one report on genetic changes for 5 genes (TP53, SF3B1, NOTCH1, MYD88, and BIRC3) by Sanger sequencing in Chinese CLL. Yet studies of CLL in Asian countries using Next generation sequencing have not been reported. We aimed to characterize the genomic profiles of Korean CLL and to find out ethnic differences in somatic mutations with prognostic implications. We performed targeted sequencing for 87 gene panel using next-generation sequencing along with G-banding and fluorescent in situ hybridization (FISH) for chromosome 12, 13q14.3 deletion, 17p13 deletion, and 11q22 deletion. Overall, 36 out of 48 patients (75%) harbored at least one mutation and mean number of mutation per patient was 1.6 (range 0-6). Aberrant karyotypes were observed in 30.4% by G-banding and 66.7% by FISH. Most recurrent mutation (>10% frequency) was ATM (20.8%) followed by TP53 (14.6%), SF3B1 (10.4%), KLHL6 (8.3%), and BCOR (6.25%). Mutations of MYD88 was associated with moderate adverse prognosis by multiple comparisons (P = 0.055). Mutation frequencies of MYD88, SAMHD1, EGR2, DDX3X, ZMYM3, and MED12 showed similar incidence with Caucasians, while mutation frequencies of ATM, TP53, KLHL6, BCOR and CDKN2A tend to be higher in Koreans than in Caucasians. Especially, ATM mutation showed 1.5 fold higher incidence than Caucasians, while mutation frequencies of SF3B1, NOTCH1, CHD2 and POT1 tend to be lower in Koreans than in Caucasians. However, mutation frequencies between Caucasians and Koreans were not significantly different statistically, probably due to low number of patients. Collectively, mutational profile and adverse prognostic genes in Korean CLL were different from those of Caucasians, suggesting an ethnic difference, while profile of cytogenetic aberrations was similar to those of Caucasians.

De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries.

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants.