Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition

Abstract

We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library construction, and 96-plex sample indexing.

Figure 1

Methods for constructing in vitro fragment libraries. (a) In the conventional protocol, mechanical or endonuclease fragmentation is followed by end-polishing, A-tailing, adaptor ligation and PCR. (b) With transposase-mediated adaptor insertion, fragmentation and adaptor insertion occur in a single 5-min in vitro step, followed by PCR. For both methods, a primer-embedded sample-specific barcode can be incorporated during PCR amplification (black triangle). Dark blue: Genomic DNA. Light green: End repaired sequence. Red: A-tail. Magenta/dark green and purple/dark green: Adaptors. Mid blue/brown/orange: Transposase adaptors. Cyan/light green triangles: Endonuclease fragmentation. Grey curved dotted lines: Sonication. Grey hexagon: Transposase.

Figure 2

Schematic of steps associated with different library preparation methods. Transposase-catalyzed adaptor insertion significantly reduces the number of steps and time associated with library construction (green path).

Figure 3

Comparison of coverage bias. (a) Coverage distribution across the E. coli genome with transposase (blue), sonication (red), and endonuclease (green) methods (solid lines) and replicates (dotted lines), normalized for total sequencing depth. (b) Coverage distribution across the PA1 and CRW10 bacteriophage genomes with transposase (blue), nebulization (red), and endonuclease (green) methods (dotted lines represent replicate libraries). (c) G+C bias for E. coli was assessed by calculating G+C content of the reference in 500 bp bins and plotting the coverage in each for transposase (blue), sonication (red), and endonuclease (green) methods, all of which show an approximately equivalent bias against the extremes.

Figure 4

Insert size showing steric hindrance. (a) Insert size was generated from libraries spiked into a paired-end 101 bp run resulting in a large proportion of reads reading into the adaptor sequence. Tails of reads were then aligned to one another to discern the insert size between adaptors, resulting in a mapping-independent insert size at the lower extreme. All reads with an insert size less than 25 bp were PCR artifacts. (b) The noticeable drop below 40 bp is consistent with a model for complete saturation of transposition events on a given stretch of DNA. The roughly 110 Å transposase homodimer (grey) is bound to genomic DNA (blue), such that the core of the enzyme acts on a 9 bp region drawn out to 41 Å as well as approximately 10 additional bases of DNA flanking either side (~34 Å each) that are essentially protected from a subsequent transposase attack due to steric hindrance. Since the core region is duplicated in the process, the minimum spacing of transposition events is approximately 38 bp.

Figure 5

Sequence coverage of human and Drosophila. (a) Coverage distribution as a percentage of the genome for human (YH1) using transposase (dark blue, autosomes; light blue, sex chromosomes) and sonication [15] (down-sampled to equivalent coverage; red, autosomes; orange, sex chromosomes) methods. Poisson (no bias) distributions (gray) with λ = 12 (sex chromosomes) and λ = 24 (autosomes) are also shown. Poisson distribution is the expected if there were absolutely no bias. (b) Coverage distribution as a percentage of Drosophila autosomes using transposase (blue, down-sampled to equivalent coverage) and sonication (Drosophila Population Genomics Project (DPGP), red) methods, as well as Poisson distribution with λ = 12 (gray). (c,d) Coverage with respect to G+C content of the reference in 10 kb or 1 kb bins for (c) human (YH1) and (d) Drosophila genomes respectively, for transposase (blue) and sonication (red) methods at comparable global genomic coverage.

Figure 6

Library complexity. Library complexity for each library shown by incremental, random sampling of 50,000 reads, without replacement, and plotting (on log-log scale) the number of uniquely occurring read-pairs with respect to total number of sampled read-pairs. Species: DM, Drosophila; EC, E. coli; NA18507 and YH1, human. Methods for fragmentation: End., endonuclease; Son., sonication; Tr., transposase. Size selection ranges are given for the YH1 libraries (all these were generated using transposase. Libararies ending in "2" are replicate libraries. 100% uniqueis in gray, i.e. the distribution if there were no duplicates of any sort.

Figure 7

PCR-free reduction in G+C coverage bias and direct-from-colony coverage distribution. (a) Coverage with respect to G+C content in E. coli with and without PCR was assessed by calculating G+C content of the reference in 500 bp bins and plotting the coverage in each for transposase after PCR (left) and with the PCR-free method (right). A significant reduction in coverage bias at the extremes of G+C content is observed. (b) Coverage distribution for E. coli library prepared directly from cell lysate without purification.