Paired end adaptor oligonucleotides

Genome DNA re-sequencing was performed by IntegraGen (Evry, France). Forty-eight genomic DNA libraries were prepared using TruSeq DNA sample preparation kit (v3) followed by paired-end 100 bases massively parallel sequencing on Illumina HiSeq 2000. Briefly, 3 μg of each sample of genomic DNA were fragmented by sonication and purified to yield fragments of 400–500 bp. Paired-end adaptor oligonucleotides from Illumina were ligated on repaired A-tailed DNA fragments, then purified and enriched by PCR cycles. Each library was quantified by qPCR before equimolar pooling of the 48 libraries. The 48-plex pool was sequenced on one flowcell lane of Illumina HiSeq 2000 platform as paired-end 100 bp reads. Image analysis and base calling were performed using Illumina Real Time Analysis (RTA) Pipeline with default parameters.

In this study, we analyzed DNA size profiles using shallow WGS, which allows the accurate estimation of the number of fragments of a certain length with a resolution of 1 bp. DSP libraries were prepared with the NEBNext® Ultra™ II kit. For library preparation, a minimum of 1 ng of cf- or cirDNA was engaged without fragmentation, and recommendations from kit providers were followed. Briefly, for DSP with the NEB kit, Illumina paired-end adaptor oligonucleotides were ligated on repaired A-tailed fragments, then SPRI purified and enriched by 11 PCR cycles with UDI primers indexing, and then SPRI purified again. The SPRI purification was adjusted to keep the small fragments around 70b of insert. Finally, the libraries to be sequenced were then precisely quantified by qPCR, to ensure that the appropriate quantity was loaded on to the Illumina sequencer, in order to obtain a minimum of 1.5 million of clusters.

DNA was extracted from two cell lines (SNU_MM1393_BM and SNU_MM1393_SC). QuickGene DNA whole blood kit S (Kurabo Industries Ltd., Japan) was used to extract DNA according to the manufacturer's recommendations. For WES, we sequenced exome using the Solexa sequencing technology platform (HiSeq2000, Illumina, San Diego, CA, USA) following the manufacturer's instructions. We randomly sheared 3 ug of genomic DNA using Covaris System to generate about 150 bp inserts. The fragmented DNA was end-repaired using T4 DNA polymerase and Klenow polymerase, and Illumina paired-end adaptor-oligonucleotides were ligated to the sticky ends. We analyzed the ligation mixture by electrophoresis on an agarose gel, sliced and purified fragments with 200–250 bp sizes. Purified DNA library was hybridized with SureSelect Human All Exon V4 probes set (Agilent, Santa Clara, CA, USA) to capture 50Mb targeted exons following manufacturer's instruction. We prepared the HiSeq2000 paired-end flow cell to the manufacturer's protocol using captured exome library. Clusters of PCR colonies were then sequenced on the HiSeq2000 platform using recommended protocols from the manufacturer.