Automated Library Construction and Analysis for High-Throughput Nanopore Sequencing of SARS-CoV-2.

BACKGROUND: To support the implementation of high-throughput pipelines suitable for SARS-CoV-2 sequencing and analysis in a clinical laboratory, we developed an automated sample preparation and analysis workflow. METHODS: We used the established ARTIC protocol with approximately 400 bp amplicons sequenced on Oxford Nanopore's MinION. Sequences were analyzed using Nextclade, assigning both a clade and quality score to each sample. RESULTS: A total of 2179 samples on twenty-five 96-well plates were sequenced. Plates of purified RNA were processed within 12 h, sequencing required up to 24 h, and analysis of each pooled plate required 1 h. The use of samples with known threshold cycle (Ct) values enabled normalization, acted as a quality control check, and revealed a strong correlation between sample Ct values and successful analysis, with 85% of samples with Ct < 30 achieving a "good" Nextclade score. Less abundant samples responded to enrichment with the fraction of Ct > 30 samples achieving a "good" classification rising by 60% after addition of a post-ARTIC PCR normalization. Serial dilutions of 3 variant of concern samples, diluted from approximately Ct = 16 to approximately Ct = 50, demonstrated successful sequencing to Ct = 37. The sample set contained a median of 24 mutations per sample and a total of 1281 unique mutations with reduced sequence read coverage noted in some regions of some samples. A total of 10 separate strains were observed in the sample set, including 3 variants of concern prevalent in British Columbia in the spring of 2021. CONCLUSIONS: We demonstrated a robust automated sequencing pipeline that takes advantage of input Ct values to improve reliability.

Alternative expression analysis by RNA sequencing.

In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription-PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation.

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 +/- 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa, version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.

The Genome sequence of the SARS-associated coronavirus.

We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.

An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones.

We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.