Tracking Down Chimeric Assemblies In The TrackIt DNA Ladder Using Nanopore Sequencing

Dataset Description These files represent two different LSK114 sequencing runs on a TrackIt 1kb Plus DNA Ladder sample, and associated data analysis. July 20 2023 Flongle Run (191 Mb; 465k reads) pod5_files_2023-Jul-20_DAE_DNA_Ladder.tar.gz- raw POD5 format files called_2023-Jul-20_DAE_DNA_Ladder_duplex.bam- duplex called reads, called using dorado v4.0 with the 2023-09-22 bacterial methylation model sequence_QC_2023-Jul-20_DAE_DNA_Ladder.pdf- sequence length / quality QC plots LAST_2023-Jul-20_DAE_DNA_Ladder_reads_vs_reference.tar.gz- Alignment summary statistics from LAST mapping of reads to their associated reference lengths_summary_2023-Jul-20_DAE_DNA_Ladder.txt- Length / QC summary statistics October 12 2023 P2 Solo Run (1.95 Gb, 1.11M reads) pod5_files_2023-Oct-12_DNA-Ladder-1kbplus_fail.tar.gz- raw POD5 format files (all failed reads) pod5_files_2023-Oct-12_DNA-Ladder-1kbplus_pass_000-059.tar.gz- raw POD5 format files (passed reads, bundle #000-059) pod5_files_2023-Oct-12_DNA-Ladder-1kbplus_pass_060-119.tar.gz- raw POD5 format files (passed reads, bundle #060-119) pod5_files_2023-Oct-12_DNA-Ladder-1kbplus_pass_120-179.tar.gz- raw POD5 format files (passed reads, bundle #120-179) pod5_files_2023-Oct-12_DNA-Ladder-1kbplus_pass_180-222.tar.gz- raw POD5 format files (passed reads, bundle #180-222) called_2023-Oct-12_DNA-Ladder-1kbplus_duplex.bam- duplex called reads [October 12, 2023], called using dorado v4.0 with the 2023-09-22 bacterial methylation model sequence_QC_2023-Oct-12_DNA-Ladder-1kbplus.pdf- sequence length / quality QC plots LAST_2023-Oct-12_DNA-Ladder-1kbplus_reads_vs_reference.tar.gz- Alignment summary statistics from LAST mapping of reads to their associated reference lengths_summary_2023-Oct-12_DNA-Ladder-1kbplus.txt- Length / QC summary statistics ladder_seqs.fa- assembled DNA ladder sequences, based on simplex reads Methods Sample preparation Preparation of DNA for sequencing was carried out following the ONT Ligation Sequencing DNA V14 (SQK-LSK114) protocol, with modifications to exclude DNA repair, and keeping the sample in the same 1.5ml tube to reduce sample loss. Tris-buffered Saline (TBS) buffer preparation 1M stock of NaCl was made by adding 2.922g of NaCl into a 50 ml Falcon tube, then made up to 50 ml with MilliPore water A 50 mM TBS stock was created by adding 750 μl 1M NaCl solution to a 15 ml Falcon tube, then made up to 15 ml using Qiagen Elution Buffer (EB, i.e. 10 mM Tris-HCl at pH 8.0) The pH was confirmed to be 7.9-8