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. 2013 Jul 9:14:466.
doi: 10.1186/1471-2164-14-466.

Multiplexed Illumina sequencing libraries from picogram quantities of DNA

Sarah K Bowman  1 , Matthew D Simon, Aimee M Deaton, Michael Tolstorukov, Mark L Borowsky, Robert E Kingston
Affiliations

Multiplexed Illumina sequencing libraries from picogram quantities of DNA

Sarah K Bowman et al. BMC Genomics. .

Abstract

Background: High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps.

Results: We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment.

Conclusions: Application of this method allows whole genome studies from samples where material or yields are limiting.

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Figures

Figure 1
Figure 1
Workflow. The ratio of the volume of suspended SPRI beads to the volume of sample is indicated.
Figure 2
Figure 2
Oligonucleotide design and products of protocol. P5 and P7 are names given by Illumina to the oligo sequences that bind to the flow cell.
Figure 3
Figure 3
The modified library protocol recapitulates known regions of enrichment in H3K27me3 ChIP. Qualitative depiction of tag density and cluster enrichment. ng: data generated from 5–50 ng ChIP DNA by modENCODE. pg1, pg2: biological replicates of data generated from ~100 pg ChIP DNA for this study. The chromatin in the ng experiment is fragmented by sonication, while the chromatin in the pg experiment is fragmented by micrococcal nuclease. A: Clusters of region of enrichment on the entirety of chr3R with a significance threshold of Z-score =3 and enrichment of 2-fold or more. y-axis is 0–3 for all samples. B: Input subtracted tag density and corresponding regions of enrichment (based on M-values) in ~1000 kb of chr3R. Y-axis is 0–250 for tag density samples and 0–3 for cluster samples. chr3R: chromosome 3R. Selected genes noted in italics.
Figure 4
Figure 4
Pairwise comparison of regions of enrichment in nanogram- and picogram-scale libraries. Each panel depicts a single pairwise comparison: ng and pg1, ng and pg2, and pg1 and pg2. Coverage of each region of enrichment in one sample (color-coded in the plots) by the regions of enrichment identified in another sample was computed as described in Methods. Then, the fractions of the total number of the regions of enrichment with coverage above the specified threshold were computed and presented in plots (solid lines). Since some regions of enrichment are not reproduced at all in different libraries, the same fractions were computed for the regions that have non-zero coverage (dashed lines) to address possible bias in the analysis.
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References

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