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After selecting the option to party queue, you will wait in the hanger bay pre-match lobby until that specific friend is done with their match, then automatically join their lobby for the next match. While queuing, you can use the voice and text chat with those currently in a match, with all queued players appearing in the game channel member list for all players in said party.
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Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform.
All the platforms have library preparation protocols that involve fragmenting genomic DNA and attaching specific adapter sequences. Typically this takes somewhere between 4 and 8 hours for one sample. In addition, the Ion Torrent template preparation has a two hour emulsion PCR and a template bead enrichment step.
In order to determine whether or not the higher error rates observed with the PGM and PacBio affected their ability to call SNPs, we aligned the reads from the S. aureus genome, for which all platforms gave good sequence representation, against the reference genome of the closely related strain USA300_FPR3757 [12], and compared the SNPs called against those obtained by aligning the reference sequences of the two genomes (Figure 5 and Additional file 5: Table S6). In order to create a fair comparison we initially used the same randomly normalized 15x datasets used in our analysis of genome coverage, which according to the literature [3] is sufficient to accurately call heterozygous variants but found that that was insufficient for the PacBio datasets where a 190x coverage was used.
Accuracy of SNP detection from the S. aureus datasets generated from each platform, compared against the reference genome of its close relative S . aureus USA300_FPR3757. Both the Torrent server variant calling pipeline and SAMtools were used for Ion Torrent data; SAMtools was used for Illumina data and SMRT portal pipeline for PacBio data. A) The percentage of SNPs detected using each platform overall (blue bar), and outside of repeats, indels and mobile genetic elements (red bar). B) The number of incorrect SNP calls for each platform overall (blue bar), and outside of repeats, indels and mobile genetic elements (red bar).
Overall the rate of SNP calling was slightly higher for the Ion Torrent data than for Illumina data (chi square p value 3.15E-08), with approximately 82% of SNPs being correctly called for the PGM and 68-76% of the SNPs being detected from the Illumina data (Figure 5A). Conversely, the rate of false SNP calls was higher with Ion Torrent data than for Illumina data (Figure 5B). SNP calling from PacBio data proved more problematic, as existing tools are optimized for short-read data and not for high error-rate long-read data. We were reliant on SNPs called by the SMRT portal pipeline for this analysis. Our results showed that SNP detection from PacBio data was not as accurate as that from the other platforms, with overall only 71% of SNPs being detected and 2876 SNPs being falsely called (Additional file 5: Table S6).
Amongst the datasets obtained from the Illumina sequencers, the percentage of correct SNP calls was higher for the MiSeq (76%) than the GAIIx (70%) data than for that obtained from the HiSeq (69%), despite the same libraries being run on both MiSeq and HiSeq. The use of Nextera library preparation gave similar results with 76% of SNPs being correctly called. It should be noted that we found the inbuilt automatic variant calling inadequate on both MiSeq and PGM, with MiSeq reporter calling just 6.6% of variants and Torrent suite 1.5.1 calling only 1.4% of variants.
Whilst one would normally use higher coverage than used here for confident SNP detection (i.e., 30-40x depth), we were limited to 15x depth due to the yield of some of the platforms. Nonetheless, at least for the haploid genome, S. aureus, 15x coverage should be a reasonable quantity for SNP detection and even in the human genome, 15x coverage has been shown to be sufficient to accurately call heterozygous SNPs [3].
Variant calling is a highly subjective process; the particular software chosen as well as the specific parameters employed to make the predictions will change the results substantially. As such, the rate of both true SNP and false positive calling provided here are purely indicative and results obtained with each sequencing platform will vary. For any particular application using a specific sequencing method, optimisation of the SNP- and indel-calling algorithm would always be recommended.
SNPs were called using the default parameters for SAMtools mpileup followed by bcftools and the SAMtools vcfutils.pl varFilter script, as described on the SAMtools webpage ( ). SNPs were also called for the Ion Torrent data using the Torrent Suite variant calling parameters for SAMtools mpileup and bcftools followed by the Torrent Suite vcf_filter.pl script.
A set of reference SNPs was created by aligning the complete S. aureus USA300_FPR3757 genome sequence with a high-quality draft sequence for S. aureus TW20 using Mugsy [32]. A single contiguous whole-genome alignment was generated by extracting aligned blocks from the Mugsy output and then manually curating. In order to control for the effects of software-specific mis-mapping, we identified and removed from our alignment regions sequences corresponding to mobile genetic elements (MGEs) in the S. aureus USA300_FPR3757 genome, along with regions with no homologue in S. aureus. MGEs were manually identified from the S. aureus USA300_FPR3757 genome annotation SNPs called from the resulting alignment provided a high-quality reference set for comparison with the SNPs identified by each sequencing platform. True SNPs are those that agree with the SNPs found in this reference set.
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