SINGLE CELL GENOMICS
The single cell platform offers several library types such as single cell 3’ and 5’ libraries, single cell immune profiling (T-and B-cell receptor rearrangements), single cell feature barcode applications, single cell ATAC-Seq, single cell CNV (DNA), CITE-seq, single cell spatial transcriptomics. Currently being tested: combined single cell ATAC and RNASeq. In 2020 342 single libraries were processed across 18 different projects touching applications from cancer to neurology.
SMART-seq single cell methodology is also being developed and used in collaborations for projects which are not suitable with the usual microfluidics methods. Another advantage of SMART-seq is the single cell resolution with full length transcript coverage.
Distribution of scRNA-seq methodologies applied in 2020 using 10x Genomics Chromium.
LONG READ, SINGLE MOLECULE SEQUENCING, AND LINKED READ SEQUENCING
The AGT team works with both Oxford Nanopore Technologies (ONT) and PacBio Sequel to perform single molecule high molecular weight DNA sequencing, as well as direct RNA sequencing using ONT platforms.
Methodologies for both isolation of ultra-high molecular weight DNA have been established by applying methodologies in collaboration with SAGE Science and Circulomics, DNA-seq and RNA-seq, as well as utilization of amplicon based libraries are established. Native (direct) RNA-seq is established and subject to continuous development. A full long read RNA-seq technology review by the AGT lab is available here.
Methods to improve the sequencing yield and the basecalling accuracy past the specifications of the long-read sequencing platforms. The methods presented are the ConcatSeq (Schlecht et al., 2017), INC-seq (Li et al., 2016), R2C2 (Volden et al., 2018), and the PacBio Circular Consensus Sequencing. In the section “increase in yield,” the green boxes present at either the 50 end or the 30 end, depending on the pool of the cDNA molecules, indicate the same DNA sequence. Similarly, for the red boxes. The sequences represented from the green and red boxes are reverse complementary with each other. The same case is for the cDNA molecules and the bridging oligo in the “increase in basecalling accuracy for the ONT platform” section. In all the sections, the two strands of the cDNA molecules are indicated with different colors. Different cDNA molecules are indicated with a different pair of strand colors.
DE NOVO GENOME SEQUENCING
The AGT team performs de novo genome sequencing using state of the art technologies based on long read sequencing, 10x Genomics Linked reads and Hi-C methodology. It is also contributing to the CanSeq150 project to enable future research in biodiversity and conservation and applications in breeding and biomedicine. We are combining methods such as long read Nanopore or Pacific Biosciences sequencing with Hi-C scaffolding. To date several fish genomes as well as other species have been assembled such as the Greenland halibut, the logperch, the yellow perch, the white sturgeon and the lake trout to name a few.