Performance Comparison of Illumina and Ion Torrent Next-Generation Sequencing Platforms for 16S rRNA-Based Bacterial Community Profiling

High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common “benchtop” sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.     

Application of Ion Torrent Sequencing to the Assessment of the Effect of Alkali Ballast Water Treatment on Microbial Community Diversity

The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies.     

Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.     

Semiconductor-based sequencing of genome-wide DNA methylation states

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we developed a MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is low cost, rapid, and scalable. We applied this protocol to demonstrate MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450 BeadChip array, and accurately identifies sites of differential DNA methylation. Furthermore, we applied an integrative approach to further investigate and confirm the role of DNA methylation in alternative splicing and to profile 5mC and 5hmC variants of DNA methylation in normal human brain tissue that is localized over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions.     

Microbial 16S rRNA Ion Tag and community metagenome sequencing using the Ion Torrent (PGM) Platform

Here we demonstrate a cost effective and scalable microbial ecology sequencing platform using the Ion Torrent Personal Genome Machine (PGM). We assessed both PCR amplified 16S rRNA and shotgun metagenomic approaches and generated 100,000+ to 1,000,000+ reads using 'post-light' based sequencing technology within different sized semi-conductor chips. Further development of Golay barcoded Ion Tags allowed multiplex analyses of microbial communities with substantially reduced costs compared with platforms such as 454/GS-FLX. Using these protocols we assessed the bacterial and archaeal dynamics within covered anaerobic digesters used to treat piggery wastes. Analysis of these sequence data showed that these novel methanogenic waste treatment systems are dominated by bacterial taxa, in particular Clostridium, Synergistia and Bacteroides that were maintained as a stable community over extended time periods. Archaeal community dynamics were more stochastic with the key methanogenic taxa more difficult to resolve, principally due to the poor congruence seen between community structures generated either by nested PCR or metagenomic approaches for archaeal analyses. Our results show that for microbial community structure and function analyses, the PGM platform provides a low cost, scalable and high throughput solution for both Tag sequencing and metagenomic analyses.     

A Next Generation Semiconductor Based Sequencing Approach for the Identification of Meat Species in DNA Mixtures

The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.     

Disparities in second‐generation DNA metabarcoding results exposed with accessible and repeatable workflows

Different second‐generation sequencing technologies may have taxon‐specific biases when DNA metabarcoding prey in predator faeces. Our major objective was to examine differences in prey recovery from bat guano across two different sequencing workflows using the same faecal DNA extracts. We compared results between the Ion Torrent PGM and the Illumina MiSeq with similar library preparations and the same analysis pipeline. We focus on repeatability and provide an R Notebook in an effort towards transparency for future methodological improvements. Full documentation of each step enhances the accessibility of our analysis pipeline. We tagged DNA from insectivorous bat faecal samples, targeted the arthropod cytochrome c oxidase I minibarcode region and sequenced the product on both second‐generation sequencing platforms. We developed an analysis pipeline with a high operational taxonomic unit (OTU) clustering threshold (i.e., ≥98.5%) followed by copy number filtering to avoid merging rare but genetically similar prey into the same OTUs. With this workflow, we detected 297 unique prey taxa, of which 74% were identified at the species level. Of these, 104 (35%) prey OTUs were detected by both platforms, 176 (59%) OTUs were detected by the Illumina MiSeq system only, and 17 (6%) OTUs were detected using the Ion Torrent system only. Costs were similar between platforms but the Illumina MiSeq recovered six times more reads and four additional insect orders than did Ion Torrent. The considerations we outline are particularly important for long‐term ecological monitoring; a more standardized approach will facilitate comparisons between studies and allow faster recognition of changes within ecological communities.     

Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis

Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1 ^c.204+1G>A, an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.     

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria

Aims

Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.

Methods and Results

Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1–V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture.

Conclusions

Sequencing the V1–V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare.

Significance and Impact of the Study

We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.     

Validation of an Ion Torrent Sequencing Platform for the Detection of Gene Mutations in Biopsy Specimens from Patients with Non-Small-Cell Lung Cancer

Background

Treatment for patients with advanced non-small cell lung cancer (NSCLC) is often determined by the presence of biomarkers that predict the response to agents targeting specific molecular pathways. Demands for multiplex analysis of the genes involved in the pathogenesis of NSCLC are increasing.

Methods

We validated the Ion Torrent Personal Genome Machine (PGM) system using the Ion AmpliSeq Cancer Hotspot Panel and compared the results with those obtained using the gold standard methods, conventional PCR and Sanger sequencing. The cycleave PCR method was used to verify the results.

Results and Conclusion

The Ion Torrent PGM resulted in a similar level of accuracy in identifying multiple genetic mutations in parallel, compared with conventional PCR and Sanger sequencing; however, the Ion Torrent PGM was superior to the other sequencing methods in terms of increased ease of use, even when taking into account the small amount of DNA that was obtained from formalin-fixed paraffin embedded (FFPE) biopsy specimens.     

Predictive Efficacy of Low Burden EGFR Mutation Detected by Next-Generation Sequencing on Response to EGFR Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Carcinoma

Direct sequencing remains the most widely used method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer; however, its relatively low sensitivity limits its clinical use. The objective of this study was to investigate the sensitivity of detecting an epidermal growth factor receptor (EGFR) mutation from peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp and Ion Torrent Personal Genome Machine (PGM) techniques compared to that by direct sequencing. Furthermore, the predictive efficacy of EGFR mutations detected by PNA-LNA PCR clamp was evaluated. EGFR mutational status was assessed by direct sequencing, PNA-LNA PCR clamp, and Ion Torrent PGM in 57 patients with non-small cell lung cancer (NSCLC). We evaluated the predictive efficacy of PNA-LNA PCR clamp on the EGFR-TKI treatment in 36 patients with advanced NSCLC retrospectively. Compared to direct sequencing (16/57, 28.1%), PNA-LNA PCR clamp (27/57, 47.4%) and Ion Torrent PGM (26/57, 45.6%) detected more EGFR mutations. EGFR mutant patients had significantly longer progressive free survival (14.31 vs. 21.61 months, P = 0.003) than that of EGFR wild patients when tested with PNA-LNA PCR clamp. However, no difference in response rate to EGFR TKIs (75.0% vs. 82.4%, P = 0.195) or overall survival (34.39 vs. 44.10 months, P = 0.422) was observed between the EGFR mutations by direct sequencing or PNA-LNA PCR clamp. Our results demonstrate firstly that patients with EGFR mutations were detected more frequently by PNA-LNA PCR clamp and Ion Torrent PGM than those by direct sequencing. EGFR mutations detected by PNA-LNA PCR clamp may be as a predicative factor for EGFR TKI response in patients with NSCLC.     

Comparing whole‐genome shotgun sequencing and DNA metabarcoding approaches for species identification and quantification of pollen species mixtures

Molecular identification of mixed‐species pollen samples has a range of applications in various fields of research. To date, such molecular identification has primarily been carried out via amplicon sequencing, but whole‐genome shotgun (WGS) sequencing of pollen DNA has potential advantages, including (1) more genetic information per sample and (2) the potential for better quantitative matching. In this study, we tested the performance of WGS sequencing methodology and publicly available reference sequences in identifying species and quantifying their relative abundance in pollen mock communities. Using mock communities previously analyzed with DNA metabarcoding, we sequenced approximately 200Mbp for each sample using Illumina HiSeq and MiSeq. Taxonomic identifications were based on the Kraken k‐mer identification method with reference libraries constructed from full‐genome and short read archive data from the NCBI database. We found WGS to be a reliable method for taxonomic identification of pollen with near 100% identification of species in mixtures but generating higher rates of false positives (reads not identified to the correct taxon at the required taxonomic level) relative to rbcL and ITS2 amplicon sequencing. For quantification of relative species abundance, WGS data provided a stronger correlation between pollen grain proportion and sequence read proportion, but diverged more from a 1:1 relationship, likely due to the higher rate of false positives. Currently, a limitation of WGS‐based pollen identification is the lack of representation of plant diversity in publicly available genome databases. As databases improve and costs drop, we expect that eventually genomics methods will become the methods of choice for species identification and quantification of mixed‐species pollen samples.     

A practical guide to DNA metabarcoding for entomological ecologists

1. DNA metabarcoding is a cost-effective species identification approach with great potential to assist entomological ecologists. This review presents a practical guide to help entomological ecologists design their own DNA metabarcoding studies and ensure that sound ecological conclusions can be obtained. 2. The review considers approaches to field sampling, laboratory work, and bioinformatic analyses, with the aim of providing the background knowledge needed to make decisions at each step of a DNA metabarcoding workflow. 3. Although most conventional sampling methods can be adapted to DNA metabarcoding, this review highlights techniques that will ensure suitable DNA preservation during field sampling and laboratory storage. The review also calls for a greater understanding of the occurrence, transportation, and deposition of environmental DNA when applying DNA metabarcoding approaches for different ecosystems. 4. Accurate species detection with DNA metabarcoding needs to consider biases introduced during DNA extraction and PCR amplification, cross-contamination resulting from inappropriate amplicon library preparation, and downstream bioinformatic analyses. Quantifying species abundance with DNA metabarcoding is in its infancy, yet recent studies demonstrate promise for estimating relative species abundance from DNA sequencing reads. 5. Given that bioinformatics is one of the biggest hurdles for researchers new to DNA metabarcoding, several useful graphical user interface programs are recommended for sequence data processing, and the application of emerging sequencing technologies is discussed.     

Method for quick DNA barcode reference library construction

DNA barcoding has become one of the most important techniques in plant species identification. Successful application of this technology is dependent on the availability of reference database of high species coverage. Unfortunately, there are experimental and data processing challenges to construct such a library within a short time. Here, we present our solutions to these challenges. We sequenced six conventional DNA barcode fragments (ITS1, ITS2, matK1, matK2, rbcL1, and rbcL2) of 380 flowering plants on next‐generation sequencing (NGS) platforms (Illumina Hiseq 2500 and Ion Torrent S5) and the Sanger sequencing platform. After comparing the sequencing depths, read lengths, base qualities, and base accuracies, we conclude that Illumina Hiseq2500 PE250 run is suitable for conventional DNA barcoding. We developed a new “Cotu” method to create consensus sequences from NGS reads for longer output sequences and more reliable bases than the other three methods. Step‐by‐step instructions to our method are provided. By using high‐throughput machines (PCR and NGS), labeling PCR, and the Cotu method, it is possible to significantly reduce the cost and labor investments for DNA barcoding. A regional or even global DNA barcoding reference library with high species coverage is likely to be constructed in a few years.     

A CRISPR-based strategy for targeted sequencing in biodiversity science

Many applications in molecular ecology require the ability to match specific DNA sequences from single- or mixed-species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target-specific enrichment capabilities of CRISPR-Cas systems may offer advantages in some applications. We identified 54,837 CRISPR-Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single- and mixed-species samples, which yielded mean chloroplast sequence lengths of 2,530–11,367 bp, depending on the experiment. In comparison to mixed-species experiments, single-species experiments yielded more on-target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed-species experiments yielded sufficient data to provide ≥48-fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplast trnL-P6 marker. Prior work developed CRISPR-based enrichment protocols for long-read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short-read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR-based analyses of mixed-species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori.     

Mapping mammalian replication domains using the ion torrent semiconductor sequencing platform

ABSTRACT

Here, we show that semiconductor-based sequencing technology can be used to map mammalian replication domains, chromosomal units with similar DNA replication timing. Replicating DNA purified from mammalian cells was successfully sequenced by the Ion Torrent platform. The resultant replication domain map of mouse embryonic stem cells is comparable to those obtained by the conventional microarray-based method.     

Mathematical modeling of 16S ribosomal DNA amplification reveals optimal conditions for the interrogation of complex microbial communities with phylogenetic microarrays

MOTIVATION: Many current studies of complex microbial communities rely on the isolation of community genomic DNA, amplification of 16S ribosomal RNA genes (rDNA) and subsequent examination of community structure through interrogation of the amplified 16S rDNA pool by high-throughput sequencing, phylogenetic microarrays or quantitative PCR. RESULTS: Here we describe the development of a mathematical model aimed to simulate multitemplate amplification of 16S ribosomal DNA sample and subsequent detection of these amplified 16S rDNA species by phylogenetic microarray. Using parameters estimated from the experimental results obtained in the analysis of intestinal microbial communities with Microbiota Array, we show that both species detection and the accuracy of species abundance estimates depended heavily on the number of PCR cycles used to amplify 16S rDNA. Both parameters initially improved with each additional PCR cycle and reached optimum between 15 and 20 cycles of amplification. The use of more than 20 cycles of PCR amplification and/or more than 50 ng of starting genomic DNA template was, however, detrimental to both the fraction of detected community members and the accuracy of abundance estimates. Overall, the outcomes of the model simulations matched well available experimental data. Our simulations also showed that species detection and the accuracy of abundance measurements correlated positively with the higher sample-wide PCR amplification rate, lower template-to-template PCR bias and lower number of species in the interrogated community. The developed model can be easily modified to simulate other multitemplate DNA mixtures as well as other microarray designs and PCR amplification protocols.     

Less is more: extreme genome complexity reduction with ddRAD using Ion Torrent semiconductor technology

Massively parallel sequencing a small proportion of the whole genome at high coverage enables answering a wide range of questions from molecular evolution and evolutionary biology to animal and plant breeding and forensics. In this study, we describe the development of restriction‐site associated DNA (RAD) sequencing approach for Ion Torrent PGM platform. Our protocol results in extreme genome complexity reduction using two rare‐cutting restriction enzymes and strict size selection of the library allowing sequencing of a relatively small number of genomic fragments with high sequencing depth. We applied this approach to a common freshwater fish species, the Eurasian perch (Perca fluviatilis L.), and generated over 2.2 MB of novel sequence data consisting of ~17 000 contigs, identified 1259 single nucleotide polymorphisms (SNPs). We also estimated genetic differentiation between the DNA pools from freshwater (Lake Peipus) and brackish water (the Baltic Sea) populations and identified SNPs with the strongest signal of differentiation that could be used for robust individual assignment in the future. This work represents an important step towards developing genomic resources and genetic tools for the Eurasian perch. We expect that our ddRAD sequencing protocol for semiconductor sequencing technology will be useful alternative for currently available RAD protocols.     

Efficient and sensitive identification and quantification of airborne pollen using next‐generation DNA sequencing

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen‐allergic patients. Current pollen monitoring methods are microscope‐based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost‐effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next‐generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.     

Evaluating bias-reducing protocols for RNA sequencing library preparation

Background

Next-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol).A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure.

Results

A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The CircLig protocol resulted in less over-representation of specific sequences than the standard protocol. Over-represented sequences are more likely to be predicted to have secondary structure and to co-fold with adaptor sequences. However, use of the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) was not sufficient to reduce bias.

Conclusions

The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Ligases that function at temperatures to remove the possible influence of secondary structure on library generation may be of value, although Mth K97A is not effective in this case.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-569) contains supplementary material, which is available to authorized users.