The technology, termed “TMI-seq” is a library preparation that combines molecular barcoding of individual molecules with “tagmentation” – a process by which the TN5 transposase both fragments and adds tags to DNA. In one use of the method, RNA is reverse transcribed to integrate a molecular barcode and a partial forward and reverse sequencing primer. It is then amplified by PCR, which integrates index sequences and full forward and reverse sequencing primers to yield a full length cDNA library. One aliquot of the library is tagmented with Tn5 and the forward sequencing primer, resulting in a library of barcoded overlapping fragments focused on the 5’ region of the target sequence. A second aliquot is tagmented with Tn5 and the reverse sequencing primer, resulting in a library of barcoded overlapping fragments focused on the 3’ region of the target sequence.
Abstract:
Although Next Generation Sequencing has vastly improved sequencing throughput while reducing sequencing costs, the preparation of nucleic acid libraries for sequencing has become a bottleneck. In addition, it is difficult using short-read next-generation sequencing to assemble highly variable sequences that exceed 500 base pairs such as cDNAs derived from antibody heavy chain, antibody light chain, and T cell variable regions RNA.
Website:
Advantages:
- Fast prep and accurate assembly for short read sequencers like Illumina sequencers.
- High quality data from immune repertoire sequences
- Accurate TCR and antibody repertoire sequences
Potential Applications:
- RNA à cDNA library sequencing
- Sequencing of T Cell receptors and antibodies
Contact Information:
Name: Jeff Jackson
Email: jjackso6@ucsc.edu
Phone: (831) 459-3976