TMI-seq: Tn5 Transposase Mediated Production of Complex Libraries for Short Read Sequencing

Background

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.  

Technology Description

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. 

Applications

• RNA à cDNA library sequencing
• Sequencing of T Cell receptors and antibodies

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

Intellectual Property Information

Related Materials

• Cole et al, Highly Accurate Sequencing of Full-Length Immune Repertoire Amplicons using Tn5-Enabled and Molecular Identifier-Guided Amplicon Assembly, J Immunol 2016; 196:2902-2907 - 02/01/2016