Massive parallel sequencing (MPS) library construction was adopted by the normal MPS construction process. The difference between the BGI and Illumina platforms was that the former involved rolling amplification while the latter used PCR amplification technology. In particular, the DNBSEQ library protocol contained three steps: including making DNA nanoballs (DNBs), loading DNBs, and sequencing. Single tube long fragment read (stLFR) library construction physically breaked the DNA into fragments of about 50Kbps, and then Tn5 transposase was used for library construction, so that each identical fragment could bear the same barcode16 (link), after the ligation step, PCR was performed and the library was ready to enter any standard MPS workflow.
Large-insert single-molecule real-time circular consensus sequencing (HiFi CCS) library preparation was conducted following the Pacific Biosciences recommended protocols40 (link). In brief, a total of 60 μg genomic DNA was sheared to ~20 kb targeted size by using Covaris g-TUBEs (Covaris). Each shearing processed 10 μg input DNA and a total of 6 shearings were performed. The sheared genomic DNA was examined by Agilent 2100 Bioanalyzer DNA12000 Chip (Agilent Technologies) for size distribution and underwent DNA damage repair/end repair, blunt-end adaptor ligation followed by exonuclease digestion.
Large-insert single-molecule real-time circular consensus sequencing (HiFi CCS) library preparation was conducted following the Pacific Biosciences recommended protocols40 (link). In brief, a total of 60 μg genomic DNA was sheared to ~20 kb targeted size by using Covaris g-TUBEs (Covaris). Each shearing processed 10 μg input DNA and a total of 6 shearings were performed. The sheared genomic DNA was examined by Agilent 2100 Bioanalyzer DNA12000 Chip (Agilent Technologies) for size distribution and underwent DNA damage repair/end repair, blunt-end adaptor ligation followed by exonuclease digestion.
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