Normalized iterative barcoding and sequencing of dna collections
An iterative and sequence-based technology, applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve problems such as growth, time-consuming, and increasing NGS libraries
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example 1
[0090] Example 1: This example illustrates the application of the methods of the present invention to collections of genomic DNA (gDNA) samples from Burkholderia strains contained in 96-well culture plates. The following experiments were performed to generate high quality gDNA sequences for each Burkholderia isolate. Sample label each gDNA sample using the modified labeling reaction setup as described below:
[0091]
[0092] SEQ ID NO:1
[0093] 5'-AGACGTGTGCTCTTCCGATCTCAACCCGAACCGAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'
[0094] SEQ ID NO: 2: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'
[0095] All 96 reactions accept / are performed with the same universal reverse primer (SEQ ID NO:2) and each reaction also accepts a different sample-labeled forward primer / with a different The sample marker forward primer was performed (ie, one of 96 unique sample marker primers, an example of which is shown in SEQ ID NO: 1). Labeling and sample labeling by PCR were performed in the same rea...
example 2
[0115] This example illustrates the use of the method of the invention for deriving long combinatorial reads from a collection of plasmid clones derived from a collection of Xenopus open reading frames (ORFs). Ten (10) 96-well plates of plasmid clones were processed as follows.
[0116] Use these reaction setup conditions for sample shotgun conditioning:
[0117]
[0118] Reactions were pooled in each well of a 96-well PCR plate using a different sample-labeled forward primer (eg, SEQ ID NO: 1), but the same common reverse primer in all 96 reactions. The reaction was then incubated in a thermal cycler as follows:
[0119]
[0120] After thermal cycling is complete, pool 10 μL of sample-labeled PCR product from each well of the PCR plate into a single 1.5 mL microcentrifuge tube ( DNA LoBind) and use a 0.7× volume of XP beads were used for purification.
[0121] Using the companion PicoGreen dsDNA analysis kit Fluorescence quantification of the purified sample-la...
example 3
[0135] This example illustrates the normalization of the present invention by comparing the number of DNA sequencing reads generated from 96 DNA standards (all of equal DNA quality) to the number of reads generated from a two-fold dilution series of the same standard DNA characteristics.
[0136] The highly active Tn5 transposase was purified to a stock concentration of 35 μM as described by Picelli et al. (Genome Research 15:2033-2040, 2014).
[0137] Prepare two 96-well plates of DNA standards (2-log DNA gradient 0.1-10.0kb, NEW ENGLAND ): Control plate A (10 ng DNA standard in all 96 wells) and dilution plate B (two-fold serial dilution of DNA standard starting with 128 ng standard input DNA in all 12 wells of column H, And end with 2ng DNA standard for all 12 wells in column B. Each sample from plates A and B was independently labeled with an identifiable sequence marker using the following reaction set-up conditions:
[0138] Add the following components to each well o...
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