BACKGROUND
sequencing technology is evolving rapidly and during the course of  <dig> several new sequencing platforms were released.
of note were the ion torrent personal genome machine  and the pacific biosciences  rs that are based on revolutionary new technologies.
the ion torrent pgm “harnesses the power of semiconductor technology” detecting the protons released as nucleotides are incorporated during synthesis  <cit> .
dna fragments with specific adapter sequences are linked to and then clonally amplified by emulsion pcr on the surface of 3-micron diameter beads, known as ion sphere particles.
the templated beads are loaded into proton-sensing wells that are fabricated on a silicon wafer and sequencing is primed from a specific location in the adapter sequence.
as sequencing proceeds, each of the four bases is introduced sequentially.
if bases of that type are incorporated, protons are released and a signal is detected proportional to the number of bases incorporated.
pacbio have developed a process enabling single molecule real time  sequencing  <cit> .
here, dna polymerase molecules, bound to a dna template, are attached to the bottom of 50 nm-wide wells termed zero-mode waveguides .
each polymerase is allowed to carry out second strand dna synthesis in the presence of γ-phosphate fluorescently labeled nucleotides.
the width of the zmw is such that light cannot propagate through the waveguide, but energy can penetrate a short distance and excite the fluorophores attached to those nucleotides that are in the vicinity of the polymerase at the bottom of the well.
as each base is incorporated, a distinctive pulse of fluorescence is detected in real time.
in recent years, the sequencing industry has been dominated by illumina, who have adopted a sequencing-by-synthesis approach  <cit> , utilizing fluorescently labeled reversible-terminator nucleotides, on clonally amplified dna templates immobilized to an acrylamide coating on the surface of a glass flowcell.
the illumina genome analyzer and more recently the hiseq  <dig> have set the standard for high throughput massively parallel sequencing, but in  <dig> illumina released a lower throughput fast-turnaround instrument, the miseq, aimed at smaller laboratories and the clinical diagnostic market.
here we evaluate the output of these new sequencing platforms and compare them with the data obtained from the illumina hiseq and gaiix platforms.
table  <dig> gives a summary of the technical specifications of each of these instruments.
* all cost calculations are based on list price quotations obtained from the manufacturer and assume expected sequence yield stated.
** system price including pgm, server, onetouch and onetouch es.
*** includes two hours of cluster generation.
**** mean mapped read length includes adapter and reverse strand sequences.
subread lengths, i.e.
the individual stretches of sequence originating from the sequenced fragment, are significantly shorter.
RESULTS
sequence generation
platform specific libraries were constructed for a set of microbial genomes bordetella pertussis , salmonella pullorum , staphylococcus aureus  and plasmodium falciparum .
we routinely use these to test new sequencing technologies, as together their sequences represent the range of genomic landscapes that one might encounter.
pcr-free  <cit>  illumina libraries were uniquely barcoded, pooled and run on a miseq flowcell with paired  <dig> base reads plus a 6-base index read and also on a single lane of an illumina hiseq with paired  <dig> base reads plus an 8-base index read .
illumina libraries prepared with amplification using kapa hifi polymerase  <cit>  were run on a single lane of an illumina ga iix with paired  <dig> base reads plus an 8-base index read and on a miseq flowcell with paired  <dig> base reads plus a 6-base index read.
pcr-free libraries represent an improvement over the standard illumina library preparation method as they result in more even sequence coverage  <cit>  and are included here alongside libraries prepared with pcr in order to enable comparison to pacbio which has an amplification free workflow.
ion torrent libraries were each run on a single  <dig> chip for a  <dig> cycles generating mean read lengths of 112– <dig> bases .
standard pacbio libraries, with an average of  <dig> kb inserts, were run individually over multiple smrt cells, each using c <dig> chemistry, and providing ≥20x sequence coverage data for each genome .
the datasets generated were mapped to the corresponding reference genome as described in methods.
for a fair comparison, all sequence datasets were randomly down-sampled  to contain reads representing a 15x average genome coverage.
workflow
all the platforms have library preparation protocols that involve fragmenting genomic dna and attaching specific adapter sequences.
typically this takes somewhere between  <dig> and  <dig> hours for one sample.
in addition, the ion torrent template preparation has a two hour emulsion pcr and a template bead enrichment step.
in the battle to become the platform with the fastest turnaround time, all the manufacturers are seeking to streamline library preparation protocols.
life technologies have developed the ion xpress fragment library kit that has an enzymatic “fragmentase” formulation for shearing starting dna, thereby avoiding the labour of physical shearing and potentially enabling complete library automation.
we tested this kit on our four genomes alongside the standard library kit with physical shearing and found both to give equal genomic representation .
illumina purchased epicentre in order to package the nextera technology with the miseq.
nextera uses a transposon to shear genomic dna and simultaneously introduce adapter sequences  <cit> .
the nextera method can produce sequencing ready dna in around  <dig> minutes and gave us remarkably even genome representation  with b. pertussis and s. aureus, but produced a very biased sequence dataset from the extremely at-rich p. falciparum genome.
genome coverage and gc bias
to analyse the uniformity of coverage across the genome we tabulated the depth of coverage seen at each position of the genome.
we utilized the coverage plots described by lam et al.,  <cit>  that depict; the percentage of the genome that is covered at a given read depth, and genome coverage at different read depths respectively, for each dataset  alongside the ideal theoretical coverage that would be predicted based on poisson behaviour.
in the context of the gc-rich genome of b. pertussis, most platforms gave similar uniformity of sequence coverage, with the ion torrent data giving slightly more uneven coverage.
in the s. aureus genome the pgm performed better.
the pgm gave very biased coverage when sequencing the extremely at-rich p. falciparum genome .
this affect was also evident when we plotted coverage depth against gc content .
whilst the pacbio platform gave a sequence dataset with quite even coverage on gc and extremely at-rich contexts, it did demonstrate slight but noticeable unevenness of coverage and bias towards gc-rich sequences with the s. aureus genome.
with the gc-neutral s. pullorum genome all platforms gave equal coverage with unbiased gc representation .
the most dramatic observation from our results was the severe bias seen when sequencing the extremely at-rich genome of p. falciparum on the pgm.
the result of this was deeper than expected coverage of the gc-rich var and subtelomeric regions and poor coverage within introns and at-rich exonic segments , with approximately 30% of the genome having no sequence coverage whatsoever.
this bias was observed with libraries prepared using both enzymatic and physical shearing .
in a recent study to investigate the optimal enzyme for next generation library preparation  <cit> , we found that the enzyme used for fragment amplification during next generation library preparation can have a significant influence on bias.
we found the enzyme kapa hifi amplifies fragments with the least bias, giving even coverage, close to that obtained without amplification.
since the pgm has two amplification steps, one during library preparation and the other emulsion pcr  for template amplification, we reasoned that this might be the cause of the observed bias.
substituting the supplied platinum taq enzyme with kapa hifi for the nick translation and amplification step during library preparation profoundly reduced the observed bias .
we were unable to further improve this by use of kapa hifi for the empcr .
of the four genomes sequenced, the p. falciparum genome is the largest and most complex and contains a significant quantity of repetitive sequences.
we used p. falciparum to analyse the effect of read length versus mappability.
as the pacbio pipeline doesn’t generate a mapping quality value and to ensure a fair comparison, we remapped the reads of all technologies using the k-mer based mapper, smalt  <cit> , and then analysed coverage across the p. falciparum genome .
this data confirms the poor performance of ion torrent on the p. falciparum genome, as only 65% of the genome is covered with high quality  reads compared to ~98-99% for the other platforms.
whilst the mean mapped readlength of the pacbio reads with this genome was  <dig> bases, average subread length  is significantly less .
the short average subread length is due to preferential loading of short fragment constructs in the library and the effect of lag time  after sequencing initiation, the latter resulting in sequences near the beginning of library constructs not being reported.
as the median length of the pacbio subreads for this data set are just  <dig> bases, we compared their coverage with an equivalent amount of in silico filtered reads of > <dig> bases.
this led to a very small decrease in the percentage of bases covered.
using paired reads on the illumina miseq, however, gave a strong positive effect, with  <dig> % more coverage being observed from paired-end reads compared to single-end reads.
error rates
we observed error rates of below  <dig> % for the illumina platforms,  <dig> % for ion torrent and 13% for pacbio sequencing .
the number of error-free reads, without a single mismatch or indel, was  <dig> %,  <dig> % and 0% for, miseq, ion torrent and pacbio, respectively.
the error heatmap in figure 2a shows that the pacbio errors are distributed evenly over the chromosome.
we manually inspected the regions where ion torrent and illumina generated more errors.
illumina produced errors after long  homopolymer tracts  <cit>  .
also evident in the miseq data, were strand errors due to the ggc motif  <cit> .
following the finding that the motif ggc generates strand-specific errors, we analyzed this phenomenon in the miseq data for p. falciparum .
we observed that the error is mostly generated by gc-rich motifs, principally ggcggg.
we found no evidence for an error if the triplet after the ggc is at-rich.
other miseq datasets also showed this artifact .
in addition to this being a strand-specific issue, it appears that this is a read-specific phenomenon.
whilst there is a quality drop in the first read following these gc-rich motifs, there is a striking loss of quality in read  <dig>  where the reads have nearly half the mean quality value compared to the read  <dig> reads for gc-rich triplets that follow the ggc motif.
we could observe this low quality in read  <dig> in all our analysed illumina lanes.
for at-rich motifs the ratio is nearly  <dig> .
ion torrent didn’t generate reads at all for long  homopolymer tracts, and could not predict the correct number of bases in homopolymers > <dig> bases long.
very few errors were observed following short homopolymer stretches in the miseq data .
additionally, we observed strand-specific errors in the pgm data but were unable to associate these with any obvious motif .
snp calling
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_fpr <dig>  <cit> , and compared the snps called against those obtained by aligning the reference sequences of the two genomes .
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  <cit>  is sufficient to accurately call heterozygous variants but found that that was insufficient for the pacbio datasets where a 190x coverage was used.
overall the rate of snp calling was slightly higher for the ion torrent data than for illumina data , with approximately 82% of snps being correctly called for the pgm and 68-76% of the snps being detected from the illumina data .
conversely, the rate of false snp calls was higher with ion torrent data than for illumina data .
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  <dig> snps being falsely called .
amongst the datasets obtained from the illumina sequencers, the percentage of correct snp calls was higher for the miseq  than the gaiix  data than for that obtained from the hiseq , 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  <dig> % of variants and torrent suite  <dig> .
<dig> calling only  <dig> % of variants.
discussion
a key feature of these new platforms is their speed.
decreasing run time has clear advantages particularly within the clinical sequencing arena, but poses challenges in itself.
whilst manufacturers may state library prep times on the order of a couple of hours, these times don’t include upfront qc and library qc and quantification.
also, typical library prep times quoted usually apply to processing of only one sample; i.e., pipetting time is largely ignored.
purchasers of sequencing instruments will want to keep them running at full utilization, in order to maximize their investment and will also want to pool multiple samples on single runs for economic reasons.
to obtain maximum throughput, users must consider the whole process, potentially investing in ancillary equipment and robotics to create an automated pipeline for the preparation of large numbers of samples.
to process large numbers of samples quickly, a facility’s instrument base must be large enough to avoid sample backlogs.
with this in mind, manufacturers are seeking to develop more streamlined sample-prep protocols that will facilitate timely sample loading.
here we have tested two such developments: enzymatic fragmentation and the nextera technique.
we conclude that these methods can be very useful, but users must carefully evaluate the methods they use for their particular applications and for use with genomes of extreme base composition to avoid bias.
whilst the data generated using the ion torrent pgm platform has a higher raw error rate  than illumina data , provided there is sufficient coverage, the representation and ability to call snps is quite closely matched between these technologies with more true positives being called from pgm data but far less false positives from the illumina data.
detection of snps using pacbio data was not as accurate; the use of single-molecule sequencing to detect low level variants and quasispecies within populations remains unproven.
we have found pacbio’s long reads useful for scaffolding de novo assemblies, though our experience suggests that this is currently not fully optimized and extensive method development is still required.
interestingly, the mappability didn’t increase significantly with longer reads, although a beneficial effect was obtained from having mate-pair information.
current pacbio protocols favor the preferential loading of smaller constructs, resulting in average subread lengths that are significantly shorter than the often quoted average read lengths.
further development is therefore required to avoid having excess short fragments and adapter-dimer constructs in the library and reducing their loading efficiency into the zmws.
whilst one would normally use higher coverage than used here for confident snp detection , 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  <cit> .
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.
we sequence many isolates of the malaria parasite p. falciparum as it represents a significant health issue in developing countries; this organism leads to several million deaths per annum.
there are several active large sequencing programs  that are currently aiming to sequence thousands of clinical malaria samples.
as the malaria genome has a gc content of only  <dig> %  <cit> , we use it as one of our test genomes, representing a significant challenge to most sequencing technologies.
based on the present study, use of illumina sequencing technology with libraries prepared without amplification  <cit>  leads to the least biased coverage across this genome.
ion torrent semiconductor sequencing is not recommended for sequencing of extremely at-rich genomes, due to the severe coverage bias observed.
this is likely to be an artifact introduced during amplification.
therefore, avoidance of library amplification and/or empcr, or use of more faithful enzymes during empcr, may eliminate the bias.
illumina sequencing has matured to the point where a great many applications  <cit>  have been developed on the platform.
since the pgm is also a massively parallel short-read technology, many of those applications should translate well and be equally practicable.
there are a few obvious exceptions; techniques involving manipulations on the flow cell such as frt-seq  <cit>  and os-seq  <cit>  will be impossible using semiconductor sequencing.
also, the ion torrent platform currently employs fragment lengths of  <dig> or  <dig> bases; without a mate-pair type library protocol, these insert sizes are too short perhaps to enable accurate de novo assemblies such as that demonstrated using allpaths-lg for mammalian genomes using illumina data  <cit> .
conversely, illumina sequencing on the hiseq or miseq instruments requires heterogeneous base composition across the population of imaged clusters  <cit> .
in order to sequence monotemplates , it is often necessary to significantly dilute or mix the sample with a complex genomic library to enable registration of clusters.
semiconductor sequencing does not suffer this problem.
the dna-input requirements of pacbio can be prohibitory.
illumina and pgm library preparation can be performed with far less dna; the standard pgm ionexpress library prep requires just 100 ng dna and illumina sequencing has been performed from sub-nanogram quantities  <cit> .
the yield, sample-input requirements and amplification-free library prep of pacbio potentially make it unsuitable for counting applications and for applications involving significant prior enrichment such as exome sequencing  <cit>  and chip-seq  <cit> .
the pacbio platform, by virtue of its long read lengths, should however have application in de novo sequencing and may also benefit analysis of linkage of alternative splicing and in of variants across long amplicons.
furthermore, the potential for direct detection of epigenetic modifications has been demonstrated  <cit> .
finally, it should be noted that thus study represents a point in time, utilising kits and reagents available up until the end of  <dig>  ion torrent and pacific biosciences are relatively new sequencing technologies that have not had time to mature in the same way that the illumina technology has.
we anticipate that whilst some of the issues identified may be intrinsic, others will be resolved as these platforms evolve.
CONCLUSIONS
the limited yield and high cost per base currently prohibit large scale sequencing projects on the pacific biosciences instrument.
the pgm and miseq are quite closely matched in terms of utility and ease of workflow.
the decision on whether to purchase one or the other will hinge on available resources, existing infrastructure and personal experience, available finances and the type of applications being considered.

 sequencing technology is evolving rapidly and during the course of several new sequencing platforms were released. of note were the ion torrent personal genome machine and the pacific biosciences rs that are based on revolutionary new technologies. the ion torrent pgm “harnesses the power of semiconductor technology” detecting the protons released as nucleotides are incorporated during synthesis. dna fragments with specific adapter sequences are linked to and then clonally amplified by emulsion pcr on the surface of 3-micron diameter beads, known as ion sphere particles. the templated beads are loaded into proton-sensing wells that are fabricated on a silicon wafer and sequencing is primed from a specific location in the adapter sequence. as sequencing proceeds, each of the four bases is introduced sequentially. if bases of that type are incorporated, protons are released and a signal is detected proportional to the number of bases incorporated. pacbio have developed a process enabling single molecule real time sequencing. here, dna polymerase molecules, bound to a dna template, are attached to the bottom of 50 nm-wide wells termed zero-mode waveguides. each polymerase is allowed to carry out second strand dna synthesis in the presence of γ-phosphate fluorescently labeled nucleotides. the width of the zmw is such that light cannot propagate through the waveguide, but energy can penetrate a short distance and excite the fluorophores attached to those nucleotides that are in the vicinity of the polymerase at the bottom of the well. as each base is incorporated, a distinctive pulse of fluorescence is detected in real time. in recent years, the sequencing industry has been dominated by illumina, who have adopted a sequencing-by-synthesis approach, utilizing fluorescently labeled reversible-terminator nucleotides, on clonally amplified dna templates immobilized to an acrylamide coating on the surface of a glass flowcell. the illumina genome analyzer and more recently the hiseq have set the standard for high throughput massively parallel sequencing, but in illumina released a lower throughput fast-turnaround instrument, the miseq, aimed at smaller laboratories and the clinical diagnostic market. here we evaluate the output of these new sequencing platforms and compare them with the data obtained from the illumina hiseq and gaiix platforms. table  gives a summary of the technical specifications of each of these instruments. * all cost calculations are based on list price quotations obtained from the manufacturer and assume expected sequence yield stated. ** system price including pgm, server, onetouch and onetouch es. *** includes two hours of cluster generation. **** mean mapped read length includes adapter and reverse strand sequences. subread lengths, i.e. the individual stretches of sequence originating from the sequenced fragment, are significantly shorter.  sequence generation platform specific libraries were constructed for a set of microbial genomes bordetella pertussis, salmonella pullorum, staphylococcus aureus and plasmodium falciparum. we routinely use these to test new sequencing technologies, as together their sequences represent the range of genomic landscapes that one might encounter. pcr-free illumina libraries were uniquely barcoded, pooled and run on a miseq flowcell with paired base reads plus a 6-base index read and also on a single lane of an illumina hiseq with paired base reads plus an 8-base index read. illumina libraries prepared with amplification using kapa hifi polymerase were run on a single lane of an illumina ga iix with paired base reads plus an 8-base index read and on a miseq flowcell with paired base reads plus a 6-base index read. pcr-free libraries represent an improvement over the standard illumina library preparation method as they result in more even sequence coverage and are included here alongside libraries prepared with pcr in order to enable comparison to pacbio which has an amplification free workflow. ion torrent libraries were each run on a single chip for a cycles generating mean read lengths of 112– bases. standard pacbio libraries, with an average of kb inserts, were run individually over multiple smrt cells, each using c chemistry, and providing ≥20x sequence coverage data for each genome. the datasets generated were mapped to the corresponding reference genome as described in methods. for a fair comparison, all sequence datasets were randomly down-sampled to contain reads representing a 15x average genome coverage. workflow all the platforms have library preparation protocols that involve fragmenting genomic dna and attaching specific adapter sequences. typically this takes somewhere between and hours for one sample. in addition, the ion torrent template preparation has a two hour emulsion pcr and a template bead enrichment step. in the battle to become the platform with the fastest turnaround time, all the manufacturers are seeking to streamline library preparation protocols. life technologies have developed the ion xpress fragment library kit that has an enzymatic “fragmentase” formulation for shearing starting dna, thereby avoiding the labour of physical shearing and potentially enabling complete library automation. we tested this kit on our four genomes alongside the standard library kit with physical shearing and found both to give equal genomic representation. illumina purchased epicentre in order to package the nextera technology with the miseq. nextera uses a transposon to shear genomic dna and simultaneously introduce adapter sequences. the nextera method can produce sequencing ready dna in around minutes and gave us remarkably even genome representation with b. pertussis and s. aureus, but produced a very biased sequence dataset from the extremely at-rich p. falciparum genome. genome coverage and gc bias to analyse the uniformity of coverage across the genome we tabulated the depth of coverage seen at each position of the genome. we utilized the coverage plots described by lam et al., that depict; the percentage of the genome that is covered at a given read depth, and genome coverage at different read depths respectively, for each dataset alongside the ideal theoretical coverage that would be predicted based on poisson behaviour. in the context of the gc-rich genome of b. pertussis, most platforms gave similar uniformity of sequence coverage, with the ion torrent data giving slightly more uneven coverage. in the s. aureus genome the pgm performed better. the pgm gave very biased coverage when sequencing the extremely at-rich p. falciparum genome. this affect was also evident when we plotted coverage depth against gc content. whilst the pacbio platform gave a sequence dataset with quite even coverage on gc and extremely at-rich contexts, it did demonstrate slight but noticeable unevenness of coverage and bias towards gc-rich sequences with the s. aureus genome. with the gc-neutral s. pullorum genome all platforms gave equal coverage with unbiased gc representation. the most dramatic observation from our results was the severe bias seen when sequencing the extremely at-rich genome of p. falciparum on the pgm. the result of this was deeper than expected coverage of the gc-rich var and subtelomeric regions and poor coverage within introns and at-rich exonic segments, with approximately 30% of the genome having no sequence coverage whatsoever. this bias was observed with libraries prepared using both enzymatic and physical shearing. in a recent study to investigate the optimal enzyme for next generation library preparation, we found that the enzyme used for fragment amplification during next generation library preparation can have a significant influence on bias. we found the enzyme kapa hifi amplifies fragments with the least bias, giving even coverage, close to that obtained without amplification. since the pgm has two amplification steps, one during library preparation and the other emulsion pcr for template amplification, we reasoned that this might be the cause of the observed bias. substituting the supplied platinum taq enzyme with kapa hifi for the nick translation and amplification step during library preparation profoundly reduced the observed bias. we were unable to further improve this by use of kapa hifi for the empcr. of the four genomes sequenced, the p. falciparum genome is the largest and most complex and contains a significant quantity of repetitive sequences. we used p. falciparum to analyse the effect of read length versus mappability. as the pacbio pipeline doesn’t generate a mapping quality value and to ensure a fair comparison, we remapped the reads of all technologies using the k-mer based mapper, smalt, and then analysed coverage across the p. falciparum genome. this data confirms the poor performance of ion torrent on the p. falciparum genome, as only 65% of the genome is covered with high quality reads compared to ~98-99% for the other platforms. whilst the mean mapped readlength of the pacbio reads with this genome was bases, average subread length is significantly less. the short average subread length is due to preferential loading of short fragment constructs in the library and the effect of lag time after sequencing initiation, the latter resulting in sequences near the beginning of library constructs not being reported. as the median length of the pacbio subreads for this data set are just bases, we compared their coverage with an equivalent amount of in silico filtered reads of > bases. this led to a very small decrease in the percentage of bases covered. using paired reads on the illumina miseq, however, gave a strong positive effect, with % more coverage being observed from paired-end reads compared to single-end reads. error rates we observed error rates of below % for the illumina platforms, % for ion torrent and 13% for pacbio sequencing. the number of error-free reads, without a single mismatch or indel, was %, % and 0% for, miseq, ion torrent and pacbio, respectively. the error heatmap in figure 2a shows that the pacbio errors are distributed evenly over the chromosome. we manually inspected the regions where ion torrent and illumina generated more errors. illumina produced errors after long homopolymer tracts. also evident in the miseq data, were strand errors due to the ggc motif. following the finding that the motif ggc generates strand-specific errors, we analyzed this phenomenon in the miseq data for p. falciparum. we observed that the error is mostly generated by gc-rich motifs, principally ggcggg. we found no evidence for an error if the triplet after the ggc is at-rich. other miseq datasets also showed this artifact. in addition to this being a strand-specific issue, it appears that this is a read-specific phenomenon. whilst there is a quality drop in the first read following these gc-rich motifs, there is a striking loss of quality in read where the reads have nearly half the mean quality value compared to the read reads for gc-rich triplets that follow the ggc motif. we could observe this low quality in read in all our analysed illumina lanes. for at-rich motifs the ratio is nearly. ion torrent didn’t generate reads at all for long homopolymer tracts, and could not predict the correct number of bases in homopolymers > bases long. very few errors were observed following short homopolymer stretches in the miseq data. additionally, we observed strand-specific errors in the pgm data but were unable to associate these with any obvious motif. snp calling 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_fpr, and compared the snps called against those obtained by aligning the reference sequences of the two genomes. 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 is sufficient to accurately call heterozygous variants but found that that was insufficient for the pacbio datasets where a 190x coverage was used. overall the rate of snp calling was slightly higher for the ion torrent data than for illumina data, with approximately 82% of snps being correctly called for the pgm and 68-76% of the snps being detected from the illumina data. conversely, the rate of false snp calls was higher with ion torrent data than for illumina data. 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 snps being falsely called. amongst the datasets obtained from the illumina sequencers, the percentage of correct snp calls was higher for the miseq than the gaiix data than for that obtained from the hiseq, 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 % of variants and torrent suite. calling only % of variants. discussion a key feature of these new platforms is their speed. decreasing run time has clear advantages particularly within the clinical sequencing arena, but poses challenges in itself. whilst manufacturers may state library prep times on the order of a couple of hours, these times don’t include upfront qc and library qc and quantification. also, typical library prep times quoted usually apply to processing of only one sample; i.e., pipetting time is largely ignored. purchasers of sequencing instruments will want to keep them running at full utilization, in order to maximize their investment and will also want to pool multiple samples on single runs for economic reasons. to obtain maximum throughput, users must consider the whole process, potentially investing in ancillary equipment and robotics to create an automated pipeline for the preparation of large numbers of samples. to process large numbers of samples quickly, a facility’s instrument base must be large enough to avoid sample backlogs. with this in mind, manufacturers are seeking to develop more streamlined sample-prep protocols that will facilitate timely sample loading. here we have tested two such developments: enzymatic fragmentation and the nextera technique. we conclude that these methods can be very useful, but users must carefully evaluate the methods they use for their particular applications and for use with genomes of extreme base composition to avoid bias. whilst the data generated using the ion torrent pgm platform has a higher raw error rate than illumina data, provided there is sufficient coverage, the representation and ability to call snps is quite closely matched between these technologies with more true positives being called from pgm data but far less false positives from the illumina data. detection of snps using pacbio data was not as accurate; the use of single-molecule sequencing to detect low level variants and quasispecies within populations remains unproven. we have found pacbio’s long reads useful for scaffolding de novo assemblies, though our experience suggests that this is currently not fully optimized and extensive method development is still required. interestingly, the mappability didn’t increase significantly with longer reads, although a beneficial effect was obtained from having mate-pair information. current pacbio protocols favor the preferential loading of smaller constructs, resulting in average subread lengths that are significantly shorter than the often quoted average read lengths. further development is therefore required to avoid having excess short fragments and adapter-dimer constructs in the library and reducing their loading efficiency into the zmws. whilst one would normally use higher coverage than used here for confident snp detection, 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. 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. we sequence many isolates of the malaria parasite p. falciparum as it represents a significant health issue in developing countries; this organism leads to several million deaths per annum. there are several active large sequencing programs that are currently aiming to sequence thousands of clinical malaria samples. as the malaria genome has a gc content of only %, we use it as one of our test genomes, representing a significant challenge to most sequencing technologies. based on the present study, use of illumina sequencing technology with libraries prepared without amplification leads to the least biased coverage across this genome. ion torrent semiconductor sequencing is not recommended for sequencing of extremely at-rich genomes, due to the severe coverage bias observed. this is likely to be an artifact introduced during amplification. therefore, avoidance of library amplification and/or empcr, or use of more faithful enzymes during empcr, may eliminate the bias. illumina sequencing has matured to the point where a great many applications have been developed on the platform. since the pgm is also a massively parallel short-read technology, many of those applications should translate well and be equally practicable. there are a few obvious exceptions; techniques involving manipulations on the flow cell such as frt-seq and os-seq will be impossible using semiconductor sequencing. also, the ion torrent platform currently employs fragment lengths of or bases; without a mate-pair type library protocol, these insert sizes are too short perhaps to enable accurate de novo assemblies such as that demonstrated using allpaths-lg for mammalian genomes using illumina data. conversely, illumina sequencing on the hiseq or miseq instruments requires heterogeneous base composition across the population of imaged clusters. in order to sequence monotemplates, it is often necessary to significantly dilute or mix the sample with a complex genomic library to enable registration of clusters. semiconductor sequencing does not suffer this problem. the dna-input requirements of pacbio can be prohibitory. illumina and pgm library preparation can be performed with far less dna; the standard pgm ionexpress library prep requires just 100 ng dna and illumina sequencing has been performed from sub-nanogram quantities. the yield, sample-input requirements and amplification-free library prep of pacbio potentially make it unsuitable for counting applications and for applications involving significant prior enrichment such as exome sequencing and chip-seq. the pacbio platform, by virtue of its long read lengths, should however have application in de novo sequencing and may also benefit analysis of linkage of alternative splicing and in of variants across long amplicons. furthermore, the potential for direct detection of epigenetic modifications has been demonstrated. finally, it should be noted that thus study represents a point in time, utilising kits and reagents available up until the end of ion torrent and pacific biosciences are relatively new sequencing technologies that have not had time to mature in the same way that the illumina technology has. we anticipate that whilst some of the issues identified may be intrinsic, others will be resolved as these platforms evolve.  the limited yield and high cost per base currently prohibit large scale sequencing projects on the pacific biosciences instrument. the pgm and miseq are quite closely matched in terms of utility and ease of workflow. the decision on whether to purchase one or the other will hinge on available resources, existing infrastructure and personal experience, available finances and the type of applications being considered. 
in order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of microbial genomes with mean gc content ranging from to %. variant calling from pacific biosciences data was possible but higher coverage depth was required. the pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: ion torrent’s pgm, pacific biosciences’ rs and the illumina miseq. 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. here we compare the results obtained with those platforms to the performance of the illumina hiseq, the current market leader. context specific errors were observed in both pgm and miseq data, but not in that from the pacific biosciences platform.
 next generation sequencing technology has revolutionized genomic and genetic research. the pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: ion torrent’s pgm, pacific biosciences’ rs and the illumina miseq. here we compare the results obtained with those platforms to the performance of the illumina hiseq, the current market leader. in order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of microbial genomes with mean gc content ranging from to %. together, these represent a comprehensive range of genome content. here we report our analysis of that sequence data in terms of coverage distribution, bias, gc distribution, variant detection and accuracy.  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 three fast turnaround sequencers evaluated here were able to generate usable sequence. however there are key differences between the quality of that data and the applications it will support. next-generation sequencingion torrentilluminapacific biosciencesmiseqpgmsmrtbiasgenome coveragegc-richat-rich 