Abstract
Linear amplification for deep sequencing (LADS) is an amplification method that produces representative libraries for Illumina next-generation sequencing within 2 d. The method relies on attaching two different sequencing adapters to blunt-end repaired and A-tailed DNA fragments, wherein one of the adapters is extended with the sequence for the T7 RNA polymerase promoter. Ligated and size-selected DNA fragments are transcribed in vitro with high RNA yields. Subsequent cDNA synthesis is initiated from a primer complementary to the first adapter, ensuring that the library will only contain full-length fragments with two distinct adapters. Contrary to the severely biased representation of AT- or GC-rich fragments in standard PCR-amplified libraries, the sequence coverage in T7-amplified libraries is indistinguishable from that of nonamplified libraries. Moreover, in contrast to amplification-free methods, LADS can generate sequencing libraries from a few nanograms of DNA, which is essential for all applications in which the starting material is limited.
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Acknowledgements
The development of this method has been financially supported by the Netherlands Organisation for Scientific Research (ZonMw/NGI Horizon 93511023; NWO-Toptalent 021.001.011) and by the European Commission (EVIMalaR; ATLAS EU-FP7_221952). We thank E. Janssen-Megens for technical assistance, advice and operation of the GAII machine and D. van Soolingen and A. Schurch for providing Mycobacterium tuberculosis genomic DNA. Furthermore, we thank A. Salcedo-Amaya and A. Brinkman for useful discussions during the development of LADS and for critical reading of the manuscript, and we are grateful for the advice and help of all our colleagues in the molecular biology department.
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R.B. developed the concept of LADS. W.A.M.H. and R.B. established and fine-tuned the protocol. K.-J.F. provided bioinformatic support. H.G.S. supervised the project. W.A.M.H., R.B. and H.G.S. prepared and wrote the manuscript.
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Supplementary information
Supplementary Figure 1 | Schematic representation of standard and amplification-free library preparation for Illumina sequencing.
(a) In the standard Illumina protocol (www.illumina.com) end-polished and A-tailed DNA fragments are ligated with a "sparrow tail" adapter containing a short complementary region and two different sequencing primers (S1, S2) on the forward and reverse strands. After size-selection, ligated fragments are subject to 12 (or even 18) cycles of PCR amplification to increase copy number. The "extended" PCR primers are also used to incorporate the sequences that facilitate hybridization to the oligonucleotides (P5, P7) attached to the surface of the flowcell; (b) In the amplification-free protocol (ref. 14) the "sparrow tail" adapters already contain the sequence used for capturing on the surface of the flowcell. Therefore, after ligation and size selection, they can be directly used for sequencing. (TIFF 581 kb)
Supplementary Figure 2 | LADS preserves GC-rich regions of the M. tuberculosis genome.
(a-c) Screenshots of the shotgun sequencing data obtained by different library preparation methods from M. tuberculosis. 40 or 400 ng sonicated genomic DNA from SH1:NLA000700874 strain was used to prepare sequencing libraries by standard PCR-based method (40ng starting material with 12 cycles of amplification), amplification-free method (400ng starting material) or LADS (40ng starting material). After sequencing on the Illumina GAII platform, 36bp sequence reads were mapped against the reference genome (H37Rv). Coverage plots were generated from equal number (∼21 million) of unique and randomly placed non-unique reads by counting the number of tags in every 10bp window of the genome. Ratio-plots display the sequencing data obtained after amplification (PCR or T7) over the amplification-free control in log2 scale (150bp moving window smoothening has been applied). (a) genome-wide view, (b-c) “zoom-in” to regions with extreme GC-content. (TIFF 1912 kb)
Supplementary Figure 3 | Decrosslinking introduces sequence bias in NGS libraries.
Formaldehyde cross-linked, fragmented Plasmodium falciparum chromatin was decrosslinked for 4 hours at 60°C or overnight at 45°C in the presence of 0.2 or 0.5M NaCl. After purification, DNA was used for Illumina library preparation using linear T7 amplification and analyzed by qPCR for amplicons with different AT-content. (TIFF 612 kb)
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Hoeijmakers, W., Bártfai, R., Françoijs, KJ. et al. Linear amplification for deep sequencing. Nat Protoc 6, 1026–1036 (2011). https://doi.org/10.1038/nprot.2011.345
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DOI: https://doi.org/10.1038/nprot.2011.345
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