1. How many cycles you are going to run? Sequencing with transposons vs. traditional sequencing TGS Methodology 1. Perform transposition reaction 2. Transform, select and retrieve plasmid 3. Sequence the plasmid 4. Read entire plasmid sequence by using just one transposition reaction ATGCTT 3.8 kb can be sequenced in 3 days, 7.6 kb can be sequenced in 3 days => Size does NOT matter Primer walking steps 1. Perform sequencing reaction 2. Read template sequence 3. Design new primers 4. REPEAT steps 1-3 multiple times until the entire plasmid is read AT ATGC ATGCTT 3.8 kb can be sequenced in 11 days, 7.6 kb can be sequenced in 20 days => Size DOES matter

2. To meet your sequencing goals, TGS will save you time. Here’s how: Turn-around time in primer walking: Sequencing is initiated with vector primers. Subsequently, new primers must be designed on the basis of the achieved sequence. It will take approximately three days to receive the new primers. Then, the sequencing and primer design steps must be repeated several times, until the entire sequence is covered. Turn-around time in TGS The complete TGS-protocol takes three days when using PCR-product as template (mapped or unmapped). With plasmid preparations, the protocol will take four days until the entire insert is sequenced.

3. Used conditions for both systems: Sequence has been read from both directions and with enough overlapping to produce sequence that fulfils the requirements of general genebanks. Read length 600 bp in one direction/ totally 1200 bp per cycle in bi-directional reactions.