1. A Novel Approach to Eliminate Vector Background and Increase Sequencing Efficiency of cDNA. Uday Matrubutham 1, Jody Mirchandani 1, Jian Liu 1, Martin A. Gleeson 1, K. MacDonald 2, J. Asano 2, Y. Butterfield 2, N. Girn 2, S. Lee 2, T. Olson 2, P. Pandoh 2, U. Skalska 2, D. Smailus 2, L. Spence 2, Jeff Stott 2, G. Yang 2, J. Schein 2 and M. Marra 2. 1 Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008 2 Genome Sequence Centre, BC Cancer Agency, Vancouver, BC, Canada Materials Introduction Conclusion We, at Invitrogen Corporation, have developed a novel and highly efficient transposon-mediated method for generating cDNA sequences with low quantities of vector background. The cDNA sequencing templates are generated in two consecutive in vitro reactions utilizing Mu transposition (GeneJumper TM ) and att/clonase recombination (Gateway TM, Invitrogen Corporation). First, the GeneJumper TM transposon is randomly inserted into the cDNA clone. Second, the cDNA insert is transferred to a compatible Gateway TM vector by recombination. Transposon-containing cDNA recombinants are identified using antibiotic selection and sequenced bi-directionally with transposon- specific primers. This process has been applied to cDNAs cloned into pCMVSport and Gateway TM clones that have att recombination sites flanking the cloned insert DNA. This methodology was successfully evaluated for high-throughput process adaptation as part of the BC Cancer Agency Genome Sequence Centre participation in the Mammalian Gene Collection initiative. The approach reduces the number of reads required to complete cDNA sequences through reduction of reads initiated from within the cDNA vector. In addition, we have adapted the approach to the pooled clone strategy currently in use at the BCCA GSC. We anticipate that the increased efficiency provided by the method will reduce dramatically the cost of sequencing cDNA clones or other similarly sized clones. Abstract Mechanism GeneJumper TM Module (Transposition) 5x Reaction buffer, GeneJumper TM Transposon-Km + (20 ng/  l), MuA Transposase (220 ng/  l), Sterile water, Control DNA, and Sequencing primers, Seq A and Seq B Gateway TM Module (Recombination) BxP module: 5x GWCT buffer, pDONR vector (150 ng/  l), BP Clonase enzyme mix, Control insert DNA LxR module: 5x LR buffer, pDEST (150 ng/  l), LR Clonase enzyme mix, Control insert DNA Competent cells TOP10 one-shot chemical competent and electro competent cells Other Materials Heat blocks @ 30  C, 75  C and 25  C; LB agar plates with Cm 12.5 /Km 50 and Cm 12.5 /Amp 50 ; Incubator @ 37  C; Culture blocs – 48 or 96 well; Plasmid DNA isolation kit; DNA sequencing reagents and instrument Currently, a major initiative is underway in the biomedical research community to collect full-length cDNAs of expressed human and mouse genes. NIH’s Mammalian Gene Collection, The German cDNA project, Japan’s NEDO project, France’s Genoscope, and the RIKEN Mouse Gene Encyclopaedia project are few examples. There are many similar efforts in the private sector as well. The primary objective is to provide access to the characterized cDNAs to the research community. The cDNAs are characterized by sequencing to determine the structure, discover novel genes and facilitate functional annotation. Depending on the program, sequencing is accomplished by shotgun method, primer walking, transposon mediated sequencing or a combination of all three methods. In the transposon technique, sequencing templates are generated by random integration of the transposon, and sequenced bi-directionally using a set of common primers that bind to either end of the transposon. It is rapid and inexpensive compared to shotgun sequencing or primer-walking. However, to avoid initiation of sequencing from transposon inserted on the vector backbone, the templates may be mapped prior to sequencing. This process is unwieldy in large-scale process as it ensues colony PCR and gel electrophoresis. We have developed a protocol using transposition and recombination that circumvents the mapping and eliminates virtually the vector background in the random sequencing of cDNA templates. The basic features of the protocol involve two consecutive in vitro reactions utilizing GeneJumper TM transposition (Invitrogen) and att/clonase Gateway TM recombination (Invitrogen). First, the GeneJumper TM transposon is integrated into cDNA inserts and second, the transposon-containing inserts are transferred to a compatible Gateway TM vector by recombination. The transposed cDNA inserts are then selected on double antibiotic containing agar plates, specific for the transposon and the vector. When randomly picked and sequenced with primers specific for the transposon, the transposed recombinants yield the entire sequence of the cDNA inserts. This protocol is anticipated to increase the sequencing efficiency of cDNA inserts and the overall cDNA clone production process. The new protocol is suitable for cDNA inserts cloned in vectors that carry bacteriophage lambda att recombination sites, flanking the cDNA insert. Clones in pCMVSPORT, Gateway TM vectors and pcDNA-att vectors are suitable for this sequencing approach. A schematic representation of the protocol is given in Figure 1. For a complete description of Gateway TM recombination mechanism please see poster 24 by Karnaoukhova et al. Experimental details II Generation of sequencing templates of Gateway TM entry clones II Generation of sequencing templates of Gateway TM entry clones cDNA insert in Gateway TM entry clones is flanked by attL1 and attL2 recombination sites. These clones are also suitable for this method to generate cDNA sequencing templates. GeneJumper TM -Gateway TM LxR reactions were performed with a single and a pool of 10 Gateway TM entry clones. Entry clone plasmid DNA was prepared using Invitrogen’s SNAP column kit for the transposition reaction. cDNA insert in Gateway TM entry clones is flanked by attL1 and attL2 recombination sites. These clones are also suitable for this method to generate cDNA sequencing templates. GeneJumper TM -Gateway TM LxR reactions were performed with a single and a pool of 10 Gateway TM entry clones. Entry clone plasmid DNA was prepared using Invitrogen’s SNAP column kit for the transposition reaction. GeneJumper TM reaction: The following were mixed in a 1.7 ml eppendorf tube. 5x reaction buffer4 ul Entry clone DNA300 ng GJ-Cm + (20 ng/ul)1.5 ul MuA transposase1 ul QS with sterile water20 ul The reaction was performed as explained in the previous experiment. Gateway TM LxR reaction: This was performed in 1.7 ml eppendorf tube. 5x reaction buffer4 ul GeneJumper rxn9 ul pDEST vector (150 ng/ ul)1 ul TE1 ul LR Clonase enzyme mix4 ul The reagents were mixed and the reaction was performed at 25  C for 4 hours. The enzyme was inactivated with the addition of 2 ul of Proteinase K and incubation of the tube at 37  C for 10 minutes. The reaction was stored on ice until transformation. Transformation: 2 ul of the reaction was transformed into Invitrogen’s One-Shot TOP10 chemical competent cells according to standard protocol. After 1 hr incubation at 37  C, 100 ul of the transformed cells was spread plated on LB agar plates containing Cm 12..5 /Amp 50. The plate was incubated at 37  C. Diagnostic PCR: Recombinants were analyzed by colony PCR to determine if the transposon was in the insert and not on the vector backbone, on randomly picked colonies using a pair of primers specific to the vector and the ends of the transposon (Mu end primer), according to the protocol in the GeneJumper TM transposon kit manual (Invitrogen Corporation). PCR products were analyzed by electrophoresis on ethidium bromide containing 1% agarose gel. Results and Discussion STEP 1: Set up GeneJumper TM transposition reaction with pCMVSport6 or Gateway cDNA clones STEP 4: Transform an aliquot of the Gateway TM reaction into E. coli and select recombinants on double antibiotic containing LB agar STEP 2: Heat kill transposase STEP 5: Extract plasmids and sequence bi-directionally using GeneJumper TM specific universal primers GeneJumper TM - transposon vector cDNA insert MuA attB2 attB1 pCMVSport6/ Gateway Expression clone vector cDNA insert attL2 attL1 Gateway Entry clone STEP 3: Transfer transposed DNA into Gateway TM BxP or Gateway TM LxR reaction using pDONR or pDEST vector, respectively + Transposition reaction containing attB1-attB2 clones or attL1- attL2 clones + attP1attP2 pDONR vector Gateway TM BxP reaction attR1attR2 pDEST vector Gateway TM LxR reaction Transposition Recombinants with transposon at random locations on the cDNA insert and NOT on the vector backbone I Generation of sequencing templates of Gateway TM pCMV.SPORT6 clones Pools of pCMV.SPORT6 cDNA clones were processed using A) the standard GeneJumper TM transposon-mediated sequencing method and B) the novel GeneJumper TM -Gateway TM BxP transposon-recombination mediated sequencing method. The objective was to determine the effectiveness of the GeneJumper TM - Gateway TM system in reducing the amount of sequence generated from vector as compared to the standard transposon-mediated cDNA sequencing method. GeneJumper TM reaction: The following were mixed in a 1.7ml eppendorf tube. 5x GWCT buffer4 ul pCMV.SPORT6 clone DNA*0.6 to 1.0 ug GJ-Cm + (20 ng/ul)3 ul MuA transposase2 ul QS with sterile water 20 ul QS with sterile water 20 ul *single clone or pool of clones were used (see result) The reagents were thoroughly mixed and the tube was incubated at 30  C for 1 hour. The transposase was heat killed at 75  C for 10 minutes. The tube was stored on ice until the Gateway TM BxP reaction was set up. Gateway TM BxP reaction: The following were mixed in a 1.7 ml eppendorf tube. 5x TCBP buffer4 ul 5x TCBP buffer4 ul GeneJumper reaction 9 ul pDONR201 (150ng/ul)1 – 2 ul QS with TE to16 ul BP Clonase enzyme mix4 ul The reagents were mixed and the reaction was performed by incubating the tube overnight at 25  C. The enzyme was inactivated with the addition of 2 ul of Proteinase K and incubation at 37  C for 10 minutes. The reaction was stored on ice until transformation. Transformation: 2 to 4 ul of the single clone reaction were transformed into 50 ul of chemically competent TOP10 One-shot cells (Invitrogen) according to standard protocol. After addition of 250 ul of SOC and incubation of the cells at 37  C for 1 hour, 200 ul of the transformed cells was spread plated on LB agar containing Cm 12.5 / Km 50. The plate was incubated at 37  C. 1 ul of the pooled cDNA reaction was electroporated into 50 ul of DH10B cells. One ml of SOC was added and incubated at 37  C for 1 hour. 500 ul of mix was spread plated on LB agar containing Cm 12.5 / Km 50. The plate was incubated at 37  C, overnight. Plasmid preparation and DNA sequencing: Plasmid DNA was prepared using standard plasmid isolation kits and DNA sequencing was performed with ABI 377 sequencer. Library cDNA clones pooled Quality bases sequenced a Quality vector bases b Quality reads Vector only reads c Insertions into vector d A91206638558.17%4214 1934 (45.89%) 723 B83189165655.64%4207 1876 (44.59%) 715 C88233944056.27%4936 2167 (43.90%) 774 Total262629748156.70%13357 5977 (44.75%) 2212 Table 2A. Standard Transposon Sequencing (pCMV.SPORT6) Library cDNA clones pooled Quality bases sequenced a Quality vector bases e Quality reads Vector only reads c Insertions into vector d D68158696811.02%3022 57 (1.89%)0 E6698450911.20%1847 29 (1.57%)0 F6452326711.62%1137 16 (1.41%)0 Total198309474411.18%6006 102 (1.70%)0 Table 2B. GeneJumper TM -Gateway TM -BxP Sequencing (pDONR201) a Bases in the quality region of the reads as determined by Phred default settings and excluding transposon sequence. b The percentage of the total quality bases that are generated from pCMV.SPORT6 vector. c The number of reads containing 97% vector bases or greater. d Calculated as the number of instances where both reads from a transposon insertion consisted of 97% vector bases or greater. Insertions into the vector close to the insert resulting in one read < 97% vector are not included in this total. e The percentage of the total quality bases that are generated from pDONR201 vector. Generation of sequencing templates for Gateway TM pCMV.SPORT6 clones Efficiency of GeneJumper TM -Gateway TM BxP reaction: The titer of recombinants obtained with pCMV.SPORT6 clones is shown in Table 1. The average titer ranged between 1x10 3 to 2.7x 10 4 per ug of the plasmid DNA. Higher titers were obtained in electroporated samples (pooled cDNA) than in chemically transformed samples. Regardless of the method of transformation, the GeneJumper TM -Gateway TM BxP system yields adequate recombinants to completely sequence any insert cDNA. Sample Titer per ug DNA (Chemical transformation) Titer per ug DNA (Electroporation) Clone 1333371667 Clone 296247116 Pool AND12833 Pool BND27192 Pool CND11726 Table 1. Titer of Recombinants (pCMV.SPORT6) Sequence Analysis: The data in Table 2 shows that there is a substantial decrease in the amount of vector sequence generated when using the GeneJumper TM -Gateway TM BxP system. With the standard Mu transposon sequencing methodology (Table 2A), 56.70% of the sequenced bases were generated from pCMV.SPORT6 vector, and 44.75% of the reads were effectively entirely vector sequence. With the GeneJumper TM -Gateway TM system (Table 2B), 11.18% of the quality bases sequenced were generated from pDONR201 vector, and just 1.7% of the reads contained only vector sequence. It should be noted that the relative amount of vector sequence generated with the standard methodology is reduced when sequencing cDNAs cloned into vectors smaller than pCMV-SPORT6. Proportion of Vector to Insert Sequence An analysis of the proportion of vector sequence generated from the two reads primed off each transposon insertion is shown in Figure 2. With the GeneJumper TM -Gateway TM system, transposon insertions into vector are not recovered and therefore at most one read from each insertion could potentially generate exclusively vector sequence. Most insertions (59%) resulted in the generation of insert-only sequence from both reads (green circle, Figure 2A). An analysis of the proportion of vector sequence generated from the two reads primed off each transposon insertion is shown in Figure 2. With the GeneJumper TM -Gateway TM system, transposon insertions into vector are not recovered and therefore at most one read from each insertion could potentially generate exclusively vector sequence. Most insertions (59%) resulted in the generation of insert-only sequence from both reads (green circle, Figure 2A). With our standard method, transposon insertions into the vector are recovered and the consequence of this is reflected in the number of insertions that generate vector sequence as compared to the GeneJumper TM -Gateway TM system (Figure 2). As shown in Figure 2B, 34.5% of insertions generate 97% or greater vector sequence from both reads. A further 21.5% of insertions resulted in one read generating 97% or greater vector sequence and the other read generating less than 97% vector sequence. Insertions of this type are not seen with the GeneJumper TM -Gateway TM system. Only 29% of insertions generated exclusively insert sequence from both reads (green circle, Figure 2B). Figure 1. Schematic Representation of GeneJumper TM -Gateway TM Protocol Figure 4. Electrophoretic Profile of Diagnostic PCR Products Sample Titer per ug of DNA (Chemical transformation) Clone 13750 Clone 24292 Pool (10 entry clones)2967 Table 3. Titer of Recombinants (pDEST vector) Efficiency of GeneJumper TM - Gateway TM LxR Reaction: The average insert size of the Gateway TM entry clones we sequenced was 1.8 kb. The titer of recombinants obtained with these clones is shown in table 3. The high titers indicate the possibility to pool more cDNA clones (60 – 65) into one reaction to obtain sequence coverage of all the cDNA inserts. For instance, if two sequence reads from a recombinant yielded 800 bases, in theory 2.25 recombinants would be required to obtain full coverage of 1.8 kb insert cDNA. When 6 to 8 fold coverage is needed 14 to 18 recombinants should be sequenced for each cDNA insert. This converts to 900 to 1200 recombinants for a pool of 65 cDNA clones. With titer as high as 4.3x10 3, it is highly possible to obtain quality sequence reads for all the cDNA inserts from a pooled sample. The high titers indicate the possibility to pool more cDNA clones (60 – 65) into one reaction to obtain sequence coverage of all the cDNA inserts. For instance, if two sequence reads from a recombinant yielded 800 bases, in theory 2.25 recombinants would be required to obtain full coverage of 1.8 kb insert cDNA. When 6 to 8 fold coverage is needed 14 to 18 recombinants should be sequenced for each cDNA insert. This converts to 900 to 1200 recombinants for a pool of 65 cDNA clones. With titer as high as 4.3x10 3, it is highly possible to obtain quality sequence reads for all the cDNA inserts from a pooled sample. Diagnostic PCR of recombinants: Transposon insertions were mapped from randomly selected clones and the results are shown in Figure 4. 98% of the analyzed clones carried one transposon in the cDNA, based on the size of the PCR product. The clones that were negative in PCR may be deemed as PCR failures or as those that produced too small a product, due to the close proximity of PCR primers, that could not be detected on the gel. Sequencing of Gateway Entry Recombinants: Sequence analysis revealed 100% transposon insertion in the cDNA insert and 0% on the vector backbone. 1.GeneJumper TM -Gateway TM system is an efficient method to generate cDNA sequencing templates with virtually zero vector background. 2.It potentially decreases (by at least 15%) the number of sequence reads required per clone to obtain full coverage of the insert DNA. 3.Finally, it reduces the cost of sequencing and enables rapid sequencing of cDNA inserts or similarly sized DNAs. ND, not determined A. GeneJumper TM -Gateway TM Sequencing Figure 2. Proportion of Vector Sequence. Individual transposon insertions plotted as a function of the percentage of vector bases generated from each of the transposon- primed sequencing reads. The x- and y-axes indicate the percentage of vector sequence derived from each of the reads, and the z-axis indicates the number of insertions with a particular proportion of vector-derived sequence from each read. B. Using standard transposon sequencing methodology. A total of 6,411 transposon insertions were analyzed. Only 1,633 (29%) of insertions generate exclusively insert sequence from both reads (green circle). The majority of the reads 2212 (34.5%) contain 97% or greater vector bases, 2089 of which are contained within the red circle. B. Standard Sequencing A. Using the GeneJumper TM /Gateway TM system. A total of 2,771 transposon insertions were analyzed. The majority of transposon insertions (1,633 or 59%) result in the generation of insert sequence exclusively from both reads (circled). A total of 13 insertions contain a proportion of vector sequence in both reads. One of these is known to be a small-insert clone (600 bp) such that both reads from the transposon insertion extended into the vector. For the remaining 12 insertions, the two reads from each transposon assembled at opposite ends of the finished cDNA sequence, which was greater than 3 kb in all cases. We are currently investigating the source of this phenomenon.