An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform
© Fadrosh et al.; licensee BioMed Central Ltd. 2014
Received: 27 October 2013
Accepted: 16 January 2014
Published: 24 February 2014
To take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of improved methods to overcome technical limitations inherent to the sequencing platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina MiSeq platform and often generates sequences of suboptimal quality.
Here we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. We introduced a 0 to 7 bp “heterogeneity spacer” to the index sequence that allows an equal proportion of samples to be sequenced out of phase.
Our approach yields high quality sequence data from 16S rRNA gene amplicons using both 250 bp and 300 bp paired-end MiSeq protocols and provides a flexible and cost-effective sequencing option.
The development of methods to detect fastidious or non-cultivable organisms through amplification and determination of the sequence of conserved genes, or culture-independent profiling, has precipitated a revolution in biology. It was recognized decades ago that the number of microbes seen on direct staining of environmental or human samples often exceeded by many orders of magnitude the number that could be cultured (termed "the great plate-count anomaly") . Culture-independent profiling of bacterial communities relies on the amplification and sequencing of the generally considered universal 16S rRNA gene and has greatly increased appreciation for the complexity hidden in even seemingly simple microbial consortia. Advancements in next-generation sequencing technologies, in terms of throughput, sequence read length and accuracy, has had a major impact in the field by enabling large numbers of samples to be examined at greater depth. The Illumina MiSeq platform (San Diego, CA, USA) provides researchers with a scalable, high-throughput and streamlined sequencing platform to survey community composition from clinical and environmental samples. However, known limitations with the MiSeq platform associated with the sequencing of low sequence diversity samples has hampered harnessing its true potential to sequence 16S rRNA gene amplicons. The “low sequence diversity” issue arises in the first several cycles of a Miseq 16S rRNA gene amplicon sequencing run, during which successful cluster identification and phasing/pre-phasing calibration are dependent on heterogeneous base composition of targeted amplicons. Because of the nature of the 16S rRNA gene, amplicon pools are highly homogenous and are required to be co-sequenced with a heterogeneous control library, commonly phage PhiX, normally combined 1:1 with the amplicon pool. This improves the quality of the sequencing reads enough to yield a successful sequencing run (average quality value of 30 (Q30) >70%) but at the expense of having half of the sequence reads lost to a non-targeted template. Despite these limitations, the MiSeq sequencing platform’s high data yield and the 250 bp and 300 bp paired-end read (250PE and 300PE) protocols continue to be attractive to researchers. The technology enables the high resolution characterization of microbial communities with effective read lengths comparable to those obtained on the Roche/454 pyrosequencing platform (Branford, CT, USA) but for a fraction of the cost.
The most widely used 16S rRNA-based MiSeq sequencing strategies include a single- [2, 3] or a recently developed dual-indexing  approach targeting the V4 hypervariable region of the 16S rRNA gene. These strategies leverage custom 16S rRNA PCR primers that enable multiplexing of samples and direct sequencing on the MiSeq instrument, but do not fully maximize the potential, or directly address the known limitations, of the sequencing technology. The single-indexing strategy requires large numbers of barcoded primers (one per sample) and custom sequencing primers, increasing costs and limiting flexibility. The current dual-indexing approach reduces the number of primers needed, but the low diversity issue still has not been addressed. This results in sequencing reads with bases of suboptimal quality that must be removed before analysis, resulting in shorter reads and analytical challenges. Here, we address the technical limitations of the MiSeq platform for 16S rRNA gene sequencing using both 250PE and 300PE protocols, and present a cost-effective approach to generate high-quality barcoded 16S rRNA gene amplicons by leveraging dual-indexed primers with built-in heterogeneity spacers.
Methods and results
V3-V4 amplification and sequencing strategy
The 16S rRNA gene consists of nine hypervariable regions flanked by regions of more conserved sequence. To maximize the effective length of the MiSeq’s 250PE and 300PE sequencing reads, a region of approximately 469 bp encompassing the V3 and V4 hypervariable regions of the 16S rRNA gene was targeted for sequencing. This region provides ample information for taxonomic classification of microbial communities from specimens associated with human microbiome studies and was used by the Human Microbiome Project , however, the approach described could be adapted to any primer pairs.
Raw sequence reads and post-QA/QC sequence reads statistics from nine 250PE and three 300PE MiSeq runs
Pool ID (run ID)
Number of samples per pool
Raw sequence reads
QA/QC sequence reads
Reported machine error
Number of read pairs
Total number of reads
Median read length
Mean read length
(S1, S2) ± SDa
266.04 ± 7.12; 246.99 ± 23.98
250PE sequence data pre-processing
Further sequence reads processing was performed using QIIME (version 1.6.0,)  and included additional quality trimming, demultiplexing, and taxonomic assignments. QIIME quality trimming was performed using the following criteria: 1) truncate sequence reads before three consecutive low-quality bases and re-evaluate for length; 2) no ambiguous base calls; and 3) minimum sequence length of 150 bp after trimming. Between 5 and 10% of the reads were filtered out when applying these quality criteria. Paired-end reads were aligned to pre-aligned Greengene 16S rRNA gene sequences. If the two paired-end reads overlapped, the consensus sequence was generated; otherwise, they were simply stitched together. While stiching reads that do not overlap allows improved taxonomic assignments, it could introduce biases when calculating operational taxonomic units.
300PE sequence data pre-processing
A total of three pools of amplicons were sequenced using the 300PE MiSeq protocol, generating between 10.7 and 14.2 (mean 12.5) million total reads (Table 1). High-quality 300PEs (Additional file 5: Figure S2) were assembled as the reads were expected to overlap by approximately 90 bp. The analysis steps were similar to that used with the 250PE protocol (Figure 2B), with the exception that paired-end reads were assembled without preliminary quality trimming, using PANDAseq  and FLASH , as both perform error correction during assembly. Overall PANDAseq and FLASH yielded very similar results with 92.99% and 92.54% of the reads assembled, respectively. The final sequence length after barcode, heterogeneity spacer, and primer removal was 429 ± 7 bp and 429 ± 6.7 bp for PANDAseq and FLASH, respectively, with an average of approximately 11,600 reads per sample.
Sequence data analysis
The MiSeq system provides a powerful sequencing platform for the rapid, high-throughput and in-depth characterization of microbial community composition using 16S rRNA gene amplicon sequencing. Its adoption was limited due to issues inherent to the technology. Two critical software processing steps for the generation of high-quality data on the MiSeq (cluster identification and phasing/prephasing rate determination) require a balanced base composition through the initial 12 to 18 cycles of the run. Because of this requirement, low sequence diversity 16S rRNA gene amplicon libraries do not sequence well on the MiSeq and the resulting data is of significantly lower overall quality than a more random library (that is, a metagenomic library). This problem is even further compounded in samples that are dominated by one or very few types of bacteria, as is the case with most vaginal microbial communities. Further, current methods for sequencing 16S rRNA gene amplicons on the MiSeq platform require a high proportion of PhiX Control library v3 (up to 50%) to be added to the amplicon library to modulate the overall sample base composition to help facilitate a successful sequencing run.
The dual-indexing amplification strategy, combined with the heterogeneity spacer design presented here, addresses the low sequence diversity issue by providing a much more balanced base composition through the entire duration of the sequencing run, increasing the overall quality of the sequence data (Additional file 1: Figure S1 and Additional file 5: Figure S2). In designing the primer system where the first 12 bases sequenced in each read are the in-line index, it is possible to select index combinations that ensure an equal proportion of each base throughout the first 12 cycles of the run. This approach also affords for multiplexing large numbers of samples at a reduced initial investment. Here, we are able to process up to 576 samples on a single sequencing run by multiplexing 24 forward and 24 reverse primers. The addition of the 0 to 7 bp heterogeneity spacer between the index sequence and the 16S rRNA sequence allows the 16S rRNA gene portion of the amplicons from an equal proportion of samples to be sequenced out of phase, further dampening the effect of the low sequence diversity issue of the MiSeq platform (Figure 1B and Additional file 1: Figure S1C). Furthermore, this feature makes it possible to dramatically reduce the ratio of PhiX Control library (v3) to amplicon library as the overall sample base composition is more even and yields data with higher overall average quality scores. Currently, the addition of as little as ~8% PhiX (Table 1) to an amplicon pool has been tested, indicating lower amounts of PhiX control library can be used to produce reads of comparable overall quality. This improved strategy produces higher quality reads (Table 1 and Additional file 4: Table S1) with substantially more usable reads, since less sequence space is dedicated to sequencing PhiX. Moreover, we have successfully adapted our method to both 250PE and 300PE MiSeq protocol, and the sequence read statistics shown in Table 1 and Additional file 1: Figure S1 and Additional file 5: Figure S2 indicates high yield and high quality sequencing reads generated for both protocols.
We designed two separate workflows that accommodate dual-indexed sequencing reads from either 250PE or 300PE protocols. We have applied a rather strict sequence quality filtering process because error rates on Illumina sequence tend to increase towards the end of the reads, with the second read being more affected than the first (see Additional file 1: Figure S1 and Additional file 5: Figure S2). This step improved the taxonomic assignment accuracy. Paired-end reads generated using the 250PE protocol can be either concatenated or stitched depending on the degree of sequence overlap. Paired-end reads generated using the 300PE protocol are assembled with great confidence using PANDAseq or FLASH. This assembly and base correction step substantially improves sequence quality as very few sequences did not pass the quality filter and no reads contained Ns (Additional file 4: Table S1). The purpose of both workflows is to maximize the information from paired-end reads to improve taxonomic assignment, while avoiding spurious paired-end reads assembly and allowing a strict, yet more flexible quality control. The taxonomic profiles of both 250PE and 300PE MiSeq runs (Figure 2) are highly similar, reflecting the high-quality sequence reads generated with either protocols and validating the method. That said, the dataset generated with the 300PE protocol has an approximate 90 bp overlap, making this protocol a preferred and superior approach that generates high-confidence paired-end assemblies compared to the 250PE, which at most overlap by 30 bp.
Current methods for sequencing 16S rRNA gene amplicons on the MiSeq instrument use custom sequencing primers complementary to the universal 16S rRNA gene universal primer [2, 4]. This custom setup creates potential problems when trying to multiplex samples on the same run that target different regions of the 16S rRNA gene. Our “heterogeneity spacer” approach to primer design and the use of the standard Illumina HP10 and HP11 sequencing primers, in combination with 300PE MiSeq protocol, yields highly reproducible datasets, and can be easily adapted to other 16 rRNA gene variable regions and future MiSeq protocol updates. While we have not observed potential amplification biases using different heterogeneity spacer length and/or sequence, it is possible that such bias exists.
Availability of supporting data
250 base pair paired-end read
300 base pair paired-end read
polymerase chain reaction
Research reported in this publication was supported by the National Institute for Allergy and Infectious Disease of the National Institutes of Health under award number UH2AI083264, U19AI084044, R33AI078798 and R01AI089878.
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