Living Single Complete Series Torrent

November 21, 2022
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Living Single Complete Series Torrent

In this study, we sequenced four bacterial genomes: two draft genomes (B. pertussis; average coverage: 109X; 1.6 Mb; 67.2% GC), and two complete reference genomes (S. aureus; average coverage: 200X; 1.8 Mb; 64.8% GC and P. falciparum; average coverage: 200X; 16.9 Mb; 51.3% GC). We sequenced each genome with three platforms, HiSeqX Ten, PacBio RS and Ion Torrent PGM.

This is a preliminary study, we have performed an initial assessment of the effect of amplification on bacterial genome sequencing using multiple methods. Use of DNA extracted from pure culture or cultures grown in broth and supplemented with CFU/mL will increase the GC bias and should be avoided. With respect to the downstream assessment of the effect of amplification we think that there is a significant bias in the initial library that then affects the behaviour of amplifying PCR products on the platform, including biases in primer-template binding, selective amplification of specific amplicons and annealing bias. The bias we observe is similar to that described by Lazarevic [ 26 ] suggesting that it is likely to be a generic issue with PCR amplification and not one specific to Mycobacterium tuberculosis. Here we have shown a comparison of four different sequencing technologies that results in a bias favouring the amplified template and we have shown that if whole genomes are sequenced, both Amplification and Sequencing Bias [ 26 ] are likely to be contributing to the resultant genome sequence. Living single complete series torrent

Although whole-genome sequencing yields the largest volumes of sequence data, it is not yet possible to analyse a genome for all of the small insertions, deletions and single base pair substitutions (SNPs) that are necessary for evolutionary analysis. The volume of sequence generated from individual human genome project projects is thus frequently required to be annotated by alignment against a reference genome. To allow for efficient search and alignment of closely related genomes using this method, we have developed a new program, SGA (Structural Genomics Assembler), to rapidly and efficiently generate genome maps from large libraries of sequence data. SGA is an independent implementation of the method used by the PhyML program for maximum-likelihood phylogenetic analyses [ 39 ], allowing the two methods to be compared. Simulated and real datasets were used to determine the improvements in alignment quality and speed obtained by using SGA. SGA reported perfect alignment of 9.4×10 extsuperscript{5} bases of sequence from the human chromosome 22, of which 93.4 extsuperscript{+7} bases (99.8%) mapped to the human genome, in 74 minutes. This compares to the 9.3×10 extsuperscript{5} bases aligned by MAUVE in 89 minutes. In the case of reads selected randomly from the Chinese isolate Zhang, the similarity between the SGA and MAUVE alignments was 99.7% of all bases aligned. This amounts to a sensitivity and specificity of 99.

Sequencing artefacts were not identified in any of the three data sets. Intra-run sequencing errors were identified in over 1% of the PacBio data, but are not observed in the Illumina data. These were predominantly located in the ends of the contigs (that is, at the beginning and end of the chromosome). Context specific sequencing errors were identified in both the MiSeq and the Ion Torrent data. Intra-run sequencing errors identified in both of the other platforms were also found in the PacBio data.
SNPs from the PacBio reads were called using BCFtools [ 9 ] in comparison with the reference genome (accession number CP000255) using a minimum depth of 20 reads, mapping quality of 30, quality score of ≥ 30, base quality of ≥ 20, read quality of ≥ 20, and mapping quality of ≥ 40 for the variant call. SNPs from both MiSeq and Ion Torrent data were called using the BCFtools consensus caller for the default settings. Hybrid assembly of the Ion Torrent data with the PacBio data produced better results than the separate assemblies. The hybrid assembly also enabled SNP calling using the Haplotype Caller, eliminating some of the errors present in the separate assemblies. These errors were at heterozygous positions with both homozygous callers.
We chose the GC- and AT-rich genomes of B. pertussis and P. falciparum to analyse the effect of read length on the mapping and consensus calling. As sequencing depth was relatively low (averaging 150-160 reads/cell), we relied on the coverage of individual genomes as a proxy for quality control. B. pertussis had similar genome sizes for all the technologies, with an average read length of 75 bases for the Ion Torrent data and an average subread length of 1130 bases for the remaining technologies. Coverage of these genomes is given in Additional file 2 : Figure S5. P. falciparum had the lowest read length (36 bases), with an average subread length of 446 bases. As expected, very few reads of >10kb were generated by the Ion Torrent, limiting its ability to cover the extremely repetitive genome of this species. The PGM had the best coverage (98.8%), although this still only provided 17 singlets of 2kb.
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Living Single Complete Series Torrent