Ultra Model Sets 40 Al 50
we identified four previously unreported retrovirus sequences from our ultra-long reads. all were recovered with high nucleotide identity and abundance ( supplementary fig. 10, supplementary table 13 ). the highest frequency isolate is listed in supplementary table 12. these retroviral sequences are distantly related (cercopithecinae)
non-genic ultra-long reads were initially identified by using a base alignment mismatch score threshold of 10. candidate non-genic sequences were further analyzed to exclude ribosomal and rrna sequences. to assess the effect of long ultra-long read length on the ability to identify non-genic ultra-long reads, we identified the number of sequences per kilobase (kb) for ultra-long reads with insert sizes from 0.5 to 8 kb. for ultra-long reads that cover the entire transcriptome, the number of base pairs identified per kb increased with read length ( fig. 6a ) and the highest base coverage is achieved with ultra-long reads of 2 kb ( fig. 6b ). ultra-long reads of 1 kb identify over 700 bp of non-genic sequence and ultra-long reads of 2 kb identify about 2,000 bp. ultra-long reads with longer insert sizes provide higher sequence coverage, but at the expense of identifying non-genic ultra-long reads.
we searched the results for bacterial sequences. within the sets of ultra-long reads produced by nanopore sequencing, >25% of reads from human cell line gm12878 were aligned to bacterial sequences (supplementary table 8). the high frequency of bacterial alignment was likely due to contamination of the minion cell lysate by environmental bacteria, since the cell line (gm12878) was derived from a donor with no known exposure to bacterial pathogens. we selected 69 bacterial sequences that aligned with read-to-reference alignments of a minimum sequence read depth of 25 in at least one of a randomly selected set of 20 gm12878 ultra-long reads. using these data as a seed, we systematically searched for bacterial sequences in the gm12878 ultra-long reads. after removing ribosomal sequences, rrna genes and some repetitive bacterial sequences, we found that the coverage increased with read length ( fig. 6c ).
all pacbio and ont ultra-long reads supported a median of ~150x median coverage for the genome. this median coverage of the genome is consistent with the cumulative number of sequencing nodes in our sequencing library. additionally, 93.9% of the genome was covered by 1.65 million reads with >50x median coverage ( fig. 7a ). using a minimum read depth of 25 per alignment, we found an average spacing of 5.2 kb between reads that covered a particular base ( fig. 7b, supplementary figs. 11 and 12 ). over 90% of reads in our data sets had a sequence read length of >50 kb. averages of the overall read lengths of k-mer length 10, 11, 12, and 13 was approximately 6.7, 8.4, 10.3, and 14.5 kb, respectively. expected tandem repeat-like reads were identified by a comparison of maximum similarity scores of the reads to the reference genome, identifying 72,611 reads spanning repetitive regions. alignment of these reads to the reference genome identified 37.3% of the repetitive regions in the genome covered by ≥20 tandem repeats, with the highest coverage of 10.
accumulation of error over time is a concern for minion sequencing using r7 sm drives, so we wanted to ensure that the gm12878 and gm12891 genomes that we used for nanopore library construction were as free of errors as possible. we assembled a c0v split-read alignment based on the gaps/stitch tool using default settings. based on a per-base error frequency for the a and c trinucleotides of 1.1% and 0.65% respectively, as estimated by the nanopore sequencing machine, 5.6% of the reads were deemed to represent errors (fig. 6a), and were filtered out. next, we aligned all draft assemblies and the gm12891 and gm12892 reference genomes to the c0v split-read alignment using minimap2 37. we estimated that this error-correction procedure improves the assembly accuracy by at least 5%, representing a measurable increase in assembly quality. for example, the largest contig improved from 170 to 183 kb (yq11.222 to yq11.224) with a higher percent of bases aligned (93.5%) and no misassemblies of the same misassemblies observed in the previous assembly ( fig. 6b).
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