single-molecule whole genome sequencing of homo sapiens: IMR-90 primary cells
DNA was extracted from a cell pellet using Zymo DNA/RNA extraction kit (Microprep plus D7005). Genomic DNA was first fragmented by double digestion with restriction endonucleases Alu I and Mlu CI (NEB, USA), overnight at 37C. After purification using 1.5 AMPure XP beads (Beckman Coulter, USA), the fragmented DNA was further processed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). The adapter provided with the kit was replaced with custom adapter P5_HP6N. After double-sided size selection using AMPure XP beads (Beckman Coulter), resulting dumbbell-like product was quantified with the Qubit kit (Thermo Fisher Scientific) and analyzed on 2100 Bioanalyzer instrument with a High Sensitivity DNA kit (Agilent, USA). To prepare P5_HP6N adapter, 86 l of 10 M solution of oligonucleotide 5-TCTTC TACAGT NNNNNN AGATCG GAAGAG CACACG TCTGAA CTCCAG TC /ideoxyU/ ACACTC TTTCCC TACACG ACGCTC TTCCGA TCT-3 (IDT, USA) in 0.1 tris-EDTA (TE) buffer was first exposed to 95C for 5 followed by 37C for 5 to form a hairpin. Next, to fill in the extending 5-end, the self-annealed oligonucleotide was supplemented with 10 l of CutSmart buffer (NEB), 2 l of 10 mM deoxynucleotide triphosphates (dNTPs) mix (NEB) and 10 U of Klenow Fragment (3  5 exo-) (NEB) and incubated at 37C for 30. After purification with the QIAquick Nucleotide Removal Kit (QIAGEN, USA), the hairpins were digested with 10 U of HpyCH4III (NEB) for 1 hour at 37C, then purified again with the QIAquick Nucleotide Removal Kit (QIAGEN), and eluted with 100 l of EB to obtain ready-to-use adapter solution. Next, for SMM-seq library preparation, samples were diluted on the basis of assessed molar concentration. Assuming 150PE sequencing mode and 30 Gb of data per sample, the dilution coefficient (D) was calculated using the formula D = M  NA  2/1025, where M is sample concentration (pM) and NA is the Avogadro constant. Resulting suspension contains ~8 amol (5 million molecules) of DNA in 1 l. Next, 1 l of diluted sample was used as a template in pulse-RCA reaction. The pulse-RCA was performed in 20-l reaction containing 1 l of diluted sample, 1 l of P5-RCA oligo (5-GTAGGGAAAGAGTGTAGACTGGAGTTC-3), 25 U (0.5 l) of strand displacement polymerase HS (BIORON Diagnostics GmbH, Germany), 2 l of buffer, 1 l of 10 mM dNTPs mix (NEB), 0.6 l of 100 mM MgCl2, and 13.9 l of water. The pulse-RCA program was set as follows: 92C for 2 min (1); 92C for 30 s (2); 60C for 30 s (3); 65C for 150 s (4); go to (3) nine times; hold at 4C. Product of amplification reaction was purified with 1.5 AMPure XP beads and resuspended in 23 l of TE buffer. The entire volume of RC amplification was PCR-amplified in 50-l reaction volume containing 23 l of RCA product, 25 l of NEBNext Ultra II Q5 Master Mix, and 1 l of P5 and P7 dual index oligos. The PCR program was set as follows: 98C for 30 s (1); 98C for 10 s (2); 65C for 75 s (3); go to (2) eight times (4); 65C for 5 min (5); 4C forever. The PCR product was purified with 0.7X AMPure XP beads and resuspended in 30 l of TE buffer. After quantification with Qubit, samples were pooled and sequenced on an Illumina NovaSeq instrument using 150 paired-end mode. Conventional sequencing library was prepared by PCR amplification of adapter-ligated samples in 30-l reaction volume containing 11 l of undiluted ligated sample, 2 U of USER enzyme (NEB), 15 l of NEBNext Ultra II Q5 Master Mix, and 1 l of P5 and P7 dual index oligos. The PCR program was set as follows: 37C for 15 min (1); 98C for 30 s (2); 98C for 10 s (3); 65C for 75 s (4); go to (3) four times (4); 65C for 5 min (5); 4C forever. The PCR product was purified with 0.7 AMPure XP beads and resuspended in 30 l of TE buffer. After quantification with Qubit, samples were pooled and sequenced on the Illumina NovaSeq instrument using 150 paired-end mode.