Although Next Generation Sequencing has vastly improved sequencing throughput while reducing sequencing costs, 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.
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.
|United States Of America||Issued Patent||11,319,576||05/03/2022||2015-974|
|United States Of America||Issued Patent||10,280,449||05/07/2019||2015-974|
Illumina (R) sequencing, Short Read Sequencing, Transposase, Tn5, TMI-Seq, Antibody sequencing, Immune Repertoire Sequencing, cDNA libraries