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  1. ...resistance to MTX (Tarassov et al. 2008) in-frame, as well as out-of-frame as a control. Dhfr along with a HPH-resistance module (on a pAG32-DHFR1,2-3; synthesized by Synbio Tech) were PCR amplified (Kapa HiFi DNA polymerase, Kapa Biosystems) using primers that, at each end, added homology regions flanking...
  2. ...reference. However, it does require high-accuracy reads (such as Illumina or PacBio HiFi) as a reference point, and ideally, these reference reads would be orthogonal to those used for the assembly.Comparison of sequencing libraries using k-mersk-mers can also be used to identify genomic differences between...
  3. ...–associated microorganisms. To obtain coverage information, we mapped HiFi reads back to the assembly with minimap2 v2.24-r1122 (-ax map-hifi) (Li 2018), whereas for taxonomic annotation, we blasted all contigs against the NCBI nt database (BLASTN -max_target_seqs 10 -max_hsps 1 -evalue 1 × 10−25). Reads mapped...
  4. ...methods.Two leading long-read sequencing technologies currently dominate the market and have significantly impacted the genomics field (Fig. 1A): Pacific Biosciences (PacBio) HiFi and Oxford Nanopore Technologies (ONT) sequencing. While both technologies produce continuous long reads, they present...
  5. ...that using the highly accurate long-read PacBio HiFi sequencing is an effective assembly strategy for highly repetitive s. During the repeat annotation, we constructed a de novo repeat library based on our assembly using RepeatModeler (Flynn et al. 2020). Combining our custom repeat library, the Dfam...
  6. ...DNA isolation.Genome assembly and refinement of the CGC1 We first assembled HiFi reads into 602 contigs with HiCanu 2.1.1 (Nurk et al. 2020), consolidated them to 80 nonredundant contigs (Supplemental Table S2A) with purge_dups 1.2.5 (Guan et al. 2020), and further reduced them to 61 nonredundant contigs...
  7. ...Enrichment method that uses the C-terminal 6-Histidine tag present on the HiFi Cas9 nuclease to pull down Cas9-bound nontarget fragments from the sequencing pool. Through HisTag-based isolation and pulldown of Cas9-bound nontarget DNA after the Cas9 cleavage step, ACME physically reduces background DNA...
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