Trimmomatic: a flexible trimmer for Illumina sequence data

Anthony M. Bolger, Marc Lohse, Bjoern Usadel · Bioinformatics · 2014 · bioinformatics · 10.1093/bioinformatics/btu170 · record bolger2014-trimmomatic-flexible-trimmer
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Performance 5 fields · coverage 4 fields · next-generation sequencing 3 fields · BWA 2 fields · Cutadapt 2 fields · de novo assembly 2 fields · and 2 more →

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next-generation sequencing NGS read preprocessing tools Trimmomatic paired-end data poor quality technical sequences suboptimal downstream analyses FASTQ files positional relationship pair awareness singletons intermediate files read trimming and filtering read preprocessing simple mode approximate match adapters short partial adapter sequences polymerase chain reaction PCR palindrome mode adapter read-through sensitivity and specificity seed and extend local alignment global alignment 16-base fragment 64-bit integer bitwise-XOR popcount quality filtering alternatives Illumina quality score Sliding Window single weak base Maximum Information adaptive approach length threshold coverage error rate correctness probabilities pipeline-based architecture paired single multiple threads Performance phred+33 phred+64 reference-based alignment Bowtie 2 BWA de novo assembly Velvet uniquely aligned reads Maximum Information approach Sliding Window approach contig N50 size maximum contig size adapter sequence AdapterRemoval Scythe/Sickle EA-Utils Cutadapt execution time other tools strict alignment criteria poor-quality input data simple-mode seed and extend palindrome signal combination paired-read alignment bitwise seed comparison adaptive strictness coverage factor error factor combined score pipeline architecture multithreading paired-end reads alignment score read quality read length strictness setting quality stringency settings system-or-tool-building
Method, as described: ordered pair-aware preprocessing steps, seed-and-extend adapter alignment, and adaptive quality/length/error scoring

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Trimmomatic: a flexible trimmer for Illumina sequence data

Abstract

contributionclaim_typeconcepts↗concepts↗concepts↗concepts↗concepts↗unhoused_relationsconcept_edgescontent_tagsMotivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data.

Results: concepts↗content_tagsThe value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested.

Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic

Contact: usadel@bio1.rwth-aachen.de

Supplementary information: Supplementary data are available at Bioinformatics online.

1 INTRODUCTION

phenomenonconcepts↗concepts↗concepts↗concept_edgesconcept_edgesThe presence of poor quality or technical sequences such as adapters in next-generation sequencing (NGS) data can easily result in suboptimal downstream analyses.

Nonetheless, it is not trivial to precisely identify such sequences, including partial adapter sequences, while leaving valid sequence data intact ( Li et al. , 2013 ). Furthermore, given the rate with which NGS sequence data are currently being produced ( Mardis, 2008 ), the additional burden of sequence preprocessing must be kept relatively modest so as to avoid adding undue overhead to the bioinformatics pipeline.

concepts↗concepts↗unhoused_relationscontent_tags↗The preprocessing approach must also not interfere with the downstream analysis of the data. For example, NGS data often come in the form of paired-end reads, and typically, the forward and reverse reads are stored in two separate FASTQ files, which contain reads from each DNA fragment in the same order. Many downstream tools use this positional relationship between pairs, so it must be maintained when preprocessing the sequence data.

limitationsconcepts↗The wide range of available NGS library preparations combined with the range of downstream applications demand a flexible approach. It should be possible to choose a set of processing steps to be applied in a user-defined order, and ideally even allow some steps to be included more than once. In other domains, this can be achieved using a shell pipeline to combine multiple tools as required, e.g. in Newick ( Junier and Zdobnov, 2010 ). However, the need for ‘pair awareness’ makes this approach difficult to apply, as the connection between the corresponding reads in the paired files will typically be lost. Correcting this would require an additional step to reconcile the read pairs and store the ‘singleton’ reads separately. Furthermore, the processing steps would not be able to assess the read pair as a unit, which is necessary or at least advantageous in some cases.

limitationsconcepts↗The alternative approach of executing a series of tools in succession would involve the creation of intermediate files at each step, a non-trivial overhead given the data size involved, and would still require pair-awareness to be built into every tool used. These issues suggest that the typical approaches to achieve flexibility by combining multiple single-purpose tools are not optimal.

research_questionsensesThus, although many NGS read preprocessing tools exist, none of them, alone or in combination, could offer the desired flexibility and performance, and most were not designed to work on paired-end data. As a result, we developed Trimmomatic as a more flexible, pair-aware and efficient preprocessing tool, optimized for Illumina NGS data.

2 ALGORITHMS

object_of_studyconcepts↗Trimmomatic includes a variety of processing steps for read trimming and filtering, but the main algorithmic innovations are related to identification of adapter sequences and quality filtering, and are described in detail below. A list of the other processing steps is presented in the Supplementary Materials .

2.1 Removal of technical sequences

limitationscontrolsconcepts↗concepts↗concepts↗concepts↗concepts↗concepts↗unhoused_relationsunhoused_relationssensesconcept_edgescontent_tagscontent_tags↗Trimmomatic uses two approaches to detect technical sequences within the reads. The first, referred to as ‘simple mode’, works by finding an approximate match between the read and the user-supplied technical sequence. This mode has the advantage of working for all technical sequences, including adapters and polymerase chain reaction (PCR) primers, or fragments thereof. Such sequences can be detected in any location or orientation within the reads but requires a substantial minimum overlap between the read and technical sequence to prevent false-positive findings. However, short partial adapter sequences, which often occur at the ends of reads, are inherently unable to meet this minimum overlap requirement and therefore are not detectable.

concepts↗concepts↗concepts↗unhoused_relationsclaim_relationscontent_tagscontent_tags↗The second mode, referred to as ‘palindrome mode’, is specifically aimed at detecting this common ‘adapter read-through’ scenario, whereby the sequenced DNA fragment is shorter than the read length, and results in adapter contamination on the end of the reads. This is especially the case for longer read length as supported by the Miseq. Although such short fragments should normally be removed during library preparation, in practice this process is not perfectly efficient, and thus many libraries suffer from this problem to some extent. ‘Palindrome mode’ can only be used with paired-end data, but has considerable advantages in sensitivity and specificity over ‘simple’ mode.

limitationsNote that the current technical sequence identification approaches in Trimmomatic are not designed to filter or categorize data on the basis of ‘barcodes’.

2.1.1 Simple mode

mechanisms↗concepts↗claim_relationscontent_tags↗content_tags↗In simple mode, each read is scanned from the 5′ end to the 3′ end to determine if any of the user-provided adapters are present. The standard ‘seed and extend’ approach ( Li and Homer, 2010 ) is used to find initial matches between the technical sequences and the reads. The seed is not required to match perfectly, and a user-defined number of mismatches are tolerated. Based on this seed match, a local alignment is performed. If the alignment score exceeds the user-defined threshold, the aligned region plus the remainder after the alignment are removed.

Figure 1 illustrates the alignments tested for each technical sequence. The process begins with a partial overlap of the 3′ end of the technical sequence with the 5′ end of the read, as shown in (A). Testing proceeds by moving the putative contaminant toward the 3′ end of the read. In both the partial overlap (A) and complete overlap at the 5′ end (B) scenarios, the entire read will be clipped. If the contaminant is found within the read (C), the bases from the 5′ end of the read to the beginning of the alignment are retained. The testing process continues until only a partial alignment on the 3′ end of the read remains (D).

Putative sequence alignments as tested in simple mode. The alignment process begins with a partial overlap at the 5′ end of the read ( A ), increasing to a full-length 5′ overlap ( B ), followed by full overlaps at all positions ( C ) and finishes with a partial overlap at the 3′ end of the read ( D ). Note that the upstream ‘adapter’ sequence is for illustration only and is not part of the read or the aligned region

Simple mode has the advantage that it can detect any technical sequence at any location in the read, provided that the alignment is sufficiently long and the read is sufficiently accurate. However, when only a short partial match is possible, such as in scenarios (A) and (D), the contaminant may not be reliably detectable.

2.1.2 Palindrome mode

mechanisms↗controlssensescontent_tags↗As noted above, ‘palindrome mode’ is specifically optimized for the detection of ‘adapter read-through’. When ‘read-through’ occurs, both reads in a pair will consist of an equal number of valid bases, followed by contaminating sequence from the ‘opposite’ adapters. Furthermore, the valid sequence within the two reads will be reverse complements. By detecting all three of these symptoms at once, adapter read-through can be identified with high sensitivity and specificity.

mechanisms↗For performance reasons, the actual algorithm combines these three tests. The adapter sequences are prepended to their respective reads, and then the combined read-with-adapter sequences from the pair are aligned against each other. A high-scoring alignment indicates that the first parts of each read are reverse complements, while the remaining parts of the reads match the respective adapters.

The alignment is implemented using a ‘seed and extend’ approach, similar to that in simple mode. Global alignment scoring is used to ensure an end-to-end match across the entire overlap.

Figure 2 illustrates the alignments tested in palindrome mode. The process begins with an overlap between the adapters and the start of the opposite reads, as shown in (A). This alignment would detect a read pair containing no useful sequence information, which could be caused by the direct ligation of the adapters. Detection of this scenario would result in the dropping of both reads. Testing then proceeds by moving the relative positioning of the reads ‘backwards’, testing for increasingly longer valid DNA fragments, illustrated in (B). This scenario would result in the trimming of both reads as illustrated. Even when only a small fragment of the adapter is overlapping, as shown in (C), the overall alignment is easily sufficient to ensure reliable detection. The process is complete when the overlapping region no longer reaches into the adapters (D).

Putative sequence alignments as tested in palindrome mode. The alignment process begins with the adapters completely overlapping the reads ( A ) testing for immediate ‘read-through’, then proceeds by checking for later overlap ( B ), including partial adapter read-through ( C ), finishing when the overlap indicates no read-through into the adapters ( D )

The main advantage of palindrome mode is the longer alignment length, which ensures that the adapters can be reliably detected, even in the presence of read errors or where only a small number of adapter bases are present. If required, palindrome mode can be used to remove even a single adapter base, while retaining a low false-positive rate. We are aware of one other tool, AdapterRemoval ( Lindgreen, 2012 ), which independently developed a similar approach.

limitationsunhoused_relationscontent_tagsNote, however, because palindrome is limited to the detection of adapter read-through, a comprehensive strategy requires the combination of both simple and palindrome modes.

2.1.3 Alignment detection and scoring

The algorithmic approach used for technical sequence alignments is somewhat unusual, avoiding the precalculated indexes often used in NGS alignments ( Li and Homer, 2010 ).

mechanisms↗concepts↗concepts↗concepts↗concepts↗sensescontent_tagscontent_tagsInitial sequence comparisons are done using a 16-base fragment from each sequence. The 16 bases are converted to the 64-bit integer, known as the seed, using a 4-bit code for each base: A = 0001, T = 0010, C = 0100 and T = 1000. These seeds are then compared using a bitwise-XOR, which determines which bits differ between the two seeds. This will result in a 0000 code for each matching base, and a code with two 1 s for each mismatch, e.g. 0011 for an A-T mismatch, as XOR(0001,0010) = 0011. The ‘1’s within this result are then counted using the ‘popcount’ operation, and this count will be exactly twice the number of differing bases for the 16-base fragments.

If the seeds are within the user-specified distance, the full alignment scoring algorithm is used. Matching bases are scored as , which is ∼0.602, while mismatches are penalized depending on their quality score, by , which can thus vary from 0 to 4. This results in a higher penalty for bases that are believed to be highly accurate.

concepts↗unhoused_relations‘Simple’ mode aligns each read against each technical sequence, using local alignment. This is implemented by finding the highest scoring region within the alignment, and thus may omit divergent regions on the ends.

concepts↗unhoused_relations‘Palindrome’ mode aligns the forward and reverse reads, combined with their adapter sequences. It uses global alignment, which is the total alignment score of the overlapping region.

2.2 Quality filtering

concepts↗concepts↗Trimmomatic offers two main quality filtering alternatives. Both approaches exploit the Illumina quality score of each base position to determine where the read should be cut, resulting in the retention of the 5′ portion, while the sequence on the 3′ of the cut point is discarded. This fits well with typical Illumina data, which generally have poorer quality toward the 3′ end. These two approaches are described in the following sections.

2.2.1 Sliding Window quality filtering

mechanisms↗concepts↗concepts↗unhoused_relationssensescontent_tagscontent_tags↗The Sliding Window uses a relatively standard approach. This works by scanning from the 5′ end of the read, and removes the 3′ end of the read when the average quality of a group of bases drops below a specified threshold. This prevents a single weak base causing the removal of subsequent high-quality data, while still ensuring that a consecutive series of poor-quality bases will trigger trimming.

2.2.2 Maximum Information quality filtering

content_tags↗A novel alternative approach was motivated by the realization that, for many applications, the incremental value of retaining additional bases in a read is related to the read length. Intuitively, it is clear that short reads are almost worthless because they occur multiple times within the target sequence and thus they give only ambiguous information. Even at the risk of introducing errors, it is worthwhile to retain additional low-quality bases early in a read, so that the trimmed read is sufficiently long to be informative.

content_tagscontent_tags↗However, beyond a certain read length, retaining additional bases is less beneficial, and may even be detrimental. Reads of moderate length are likely to be already informative and, depending on the task at hand, can be almost as valuable as full-length reads. Therefore, the smaller potential benefit of retaining additional bases must be balanced against the increasing risk of retaining errors, which could cause the existing read value to be lost.

mechanisms↗As such, it is worthwhile for the trimming process to become increasingly strict as it progresses through the read, rather than to apply a fixed quality threshold. To the best of our knowledge, this approach has not been applied in any existing tools.

concepts↗concepts↗unhoused_relationssensescontent_tags↗The ‘Maximum Information’ quality filtering approach implements this adaptive approach. It uses a combination of three factors to determine how much of each read should be retained.

mechanisms↗concepts↗content_tags↗content_tags↗The first factor models the ‘length threshold’ concept, whereby a read must be of at least a minimal length to be useful for the downstream application. As described above, very short reads have little value, as they are too ambiguous to be informative. On the other hand, most long reads can be mapped to few locations in the target sequence. If they cannot be uniquely mapped, because of them originating in a repetitive region, it is unlikely that a small number of additional bases will resolve this. For reads between these extremes, the marginal benefit of a small number of additional bases is considerable, as these extra bases may make the difference between an ambiguous and an informative read.

A logistic curve was chosen to implement this scoring behavior, as it gives a relatively flat score for extreme values, while providing a steep transition around the user-specified threshold point. Given a target length t , the putative trimming to length l would give a length threshold score:

mechanisms↗concepts↗content_tags↗content_tags↗The second factor models ‘coverage’, and provides a linear score based on retained sequence length:

This reflects that, given reasonably high-accuracy bases, a longer read contains more information that is useful for most applications.

mechanisms↗concepts↗content_tags↗content_tags↗The final factor models the ‘error rate’, and uses the error probabilities from the read quality scores to determine the accumulated likelihood of errors over the read. To calculate this score, we simply take the product of the probabilities that each base is correct, giving:

concepts↗The correctness probabilities P corr of each base are calculated from the sequence quality scores. The error score typically begins as a high score at the start of the read, and depending on the read quality, typically drops rapidly at some point during the read.

content_tagscontent_tags↗The Maximum Information algorithm determines the combined score of the three factors for each possible trimming position, and the best combined score determines how much of the read to trim. A user-specified strictness setting s , which can be set between 0 and 1, controls the balance between the ‘coverage’ factor (maximal for s = 0) and the ‘error rate’ factor (maximal for s = 1). This gives the following overall formula:

mechanisms↗Figure 3 illustrates how the three factors are combined into a single score. The peak score is then used to determine the point where the read is trimmed.

How Maximum Information mode combines uniqueness, coverage and error rate to determine the optimal trimming point

3 IMPLEMENTATION

method↗mechanisms↗concepts↗concepts↗concepts↗unhoused_relationssensescontent_tags↗Trimmomatic uses a pipeline-based architecture, allowing individual ‘steps’ (adapter removal, quality filtering, etc.) to be applied to each read/read pair, in the order specified by the user. Each step can choose to work on the reads in isolation, or work on the combined pair, as appropriate. The tool tracks read pairing and stores ‘paired’ and ‘single’ reads separately.

A full list of the additional trimming and filtering steps is given in the Supplementary Materials and the online manual.

3.1 Convenience features

Input and output files can be specified individually on the command line, but for paired-end mode, where two similarly named input and four similarly named output files are often used, a ‘template’ name can be given instead of the input and/or output files. This template is automatically expanded to give the complete set of files needed. See Supplementary Materials for more details.

Compressed input and output are supported using either gzip or bzip2 formats. Compression/decompression is applied automatically when the appropriate file extensions are used, e.g. gz or bz2.

mechanisms↗concepts↗concepts↗unhoused_relationsPerformance can be improved using multiple threads if multiple CPU cores are available. The number of threads to use can be specified by the user or will be determined automatically if unspecified.

concepts↗concepts↗Trimmomatic supports sequence quality data in both standard (phred+33) and Illumina ‘legacy’ formats (phred+64), and can also convert between these formats if required. The quality format is determined automatically if not specified by the user.

limitationsThe trimming status of each read can optionally be written to a log file. This is intended to help tune the choice of processing parameters used, but because it has a significant performance impact, it is not recommended unless needed.

4 RESULTS

concepts↗concepts↗concepts↗concepts↗concepts↗claim_relationsclaim_relationsTo illustrate the value of data preprocessing, we evaluated two different scenarios: reference-based alignment using Bowtie 2 ( Langmead and Salzberg, 2012 ) and BWA ( Li and Durbin, 2009 ) against the Escherichia coli K-12/MG1655 reference (NCBI sequence NC_000913.2 ), and de novo assembly using Velvet ( Zerbino and Birney, 2008 ), on public E.coli K-12/MG1655 datasets (SRA datasets SRX131047 and SRR519926), as described in the Supplementary Methods .

4.1 Reference-based alignment

content_tagsDataset 1 (SRX131047) represents a typical Illumina library, sequenced on the HiSeq 2000 using 2 × 100 bp reads. Quality checking with FastQC revealed a notable quality drop in many reads after cycle 75 in both but did not report a high level of adapter contamination.

concepts↗concepts↗concepts↗unhoused_relationsconcept_edgescontent_tagscontent_tagscontent_tags↗In the reference-based scenario, preprocessing increased the number of uniquely aligned reads from dataset 1, as seen in the first portion of Table 1 . Filtering for both adapters and quality achieves the best result, and quality trimming is especially important when alignment settings are strict. The Maximum Information approach outperforms the Sliding Window approach in both cases, with a wider margin when the alignment mode is strict.

Results of alignment of raw data and data trimmed by Trimmomatic from both datasets

content_tags↗Notably, the optimal results for strict alignment and tolerant alignment were found using widely different quality stringency settings. (See Supplementary Results for more details.)

controlsTo validate these results with an alternative aligner, we repeated the experiment using BWA. Although the alignment counts differ, because of slight differences between the tools in the settings or algorithms, the overall trend is similar. The best results are again achieved when filtering for both adapters and quality, as shown in the second part of Table 1 .

content_tagsDataset 2 (SRR519926) is a 2 × 250 bp run, sequenced on an MiSeq. Although read quality is high at the start of each forward read, the longer read length allows more opportunity for errors to accumulate in the lower quality final 60–70 bases of each read. Furthermore, the reverse read has notably poorer quality, with quality dropping significantly by approximately base 120. These quality issues can be seen clearly in the FastQC plots, shown in the Supplementary Figure S1 , compared with the much higher average quality of the post-filtered data, as shown in Supplementary Figure S2 .

content_tagscontent_tagscontent_tagsNot surprisingly, trimming is even more critical to achieving acceptable alignment rates with these data. The final part of Table 1 shows that <1.5% of the reads align in strict mode, which requires a perfect match, while just 7% of the reads can be aligned when allowing for one mismatch. Even with the liberal default settings, allowing nine mismatches, <25% (197 933 reads) can be aligned. However, after trimming, almost 78% of the reads align perfectly.

4.2 De novo assembly

concepts↗concepts↗concepts↗concept_edgesconcept_edgescontent_tagscontent_tagscontent_tags↗content_tags↗Both datasets also showed considerable improvement in a de novo assembly scenario. For the first dataset, the contig N50 size increased by 58% (95 389 versus 60 370 bp) after preprocessing, while the maximum contig size improved by ∼28%. Also, the assembly from unfiltered data contained a 34-bp perfect match to an adapter sequence, while no adapters were found in the filtered assemblies.

content_tagscontent_tagsThe second dataset showed even greater benefits after trimming, with ∼77% improvement in N50 contig size (177 880 versus 100 662 bp) and ∼55% increase in maximum contig size. Perhaps surprisingly, no adapter sequences were found in the assembly of the untrimmed version of this dataset.

4.3 Comparison with existing tools

concepts↗concepts↗concepts↗concepts↗concepts↗unhoused_relationsWe also compared the performance of Trimmomatic with a variety of existing adapter and quality filtering tools in similar referenced-based scenarios, as described in the Supplementary Methods . The tools selected were AdapterRemoval ( Lindgreen, 2012 ), and Scythe/Sickle ( https://github.com/najoshi/ ), which fully support paired-end data and EA-Utils ( Aronesty, 2013 ), which maintains read pairing but loses singletons (reads whose mate has been filtered). Additionally, the single-end tools Cutadapt ( Martin, 2011 ), Fastx-Toolkit ( http://hannonlab.cshl.edu/fastx_toolkit ) and Reaper ( http://www.ebi.ac.uk/∼stijn/reaper ) were included.

limitationsWe filtered and aligned using paired-end mode for those tools that support it, but we used single-end mode as a fallback where necessary. In practice, ignoring pairing will result in suboptimal alignments but was done here in the interest of making the output of all tools comparable.

controlscontent_tagsTable 2 shows the output of the various tools aligned using Bowtie 2 in both tolerant and strict alignment settings. The top portion of this table, which shows the results using a tolerant alignment, suggests that the best tools perform almost identically in terms of output quality, with <20 000 reads separating the top three, and most tools within ∼1% of the best. However, given that the unfiltered data show a difference of just 1.5%, the narrowness of the result is likely due to the relatively low rate of adapter contamination in this dataset, the high average read quality and the tolerant alignment settings used.

Results of Bowtie2 alignment of dataset 1 showing raw data and the trimmed data by each tool

limitationsconcepts↗content_tags↗The execution time varies widely, with EA-Utils leading, Trimmomatic following closely, while the remaining tools require considerably longer time. However, the testing methodology, using the median of 3 runs on a relatively small dataset, allows the entire dataset to be cached. In practice, it is likely that at least the faster tools will be limited by IO performance. The individual execution times for each run are shown in Supplementary Table S4 .

The equivalent alignment using strict mode, shown in the bottom part of the table, more clearly differentiates the tools, with the margin between Trimmomatic in Maximum Information mode and the alternatives widening considerably.

Alignments of the same dataset using BWA painted a broadly similar picture, as shown in the top half of Table 3 , although the difference between strict and tolerant mode is not so strong. Again, Maximum Information mode appears to outperform by a greater margin for stricter alignments.

Results of strict and tolerant BWA alignments of the raw data and trimmed data from each tool (using both quality modes for Trimmomatic) from both datasets

content_tagsThe results of dataset 2, shown in the lower half of Table 3 , rank many of the tools differently, with AdapterRemoval dropping significantly in the ranking. Trimmomatic remains the best performer, especially in Maximum Information mode, but Cutadapt becomes the closest challenger. Reaper was unable to process this dataset, perhaps because of the long read length.

5 DISCUSSION

5.1 The need for read preprocessing

limitationssensescontent_tagsWe have illustrated the advantages of NGS data preprocessing in both reference-based and de novo assembly applications. For high-quality datasets, in reference-based applications, the benefits of preprocessing seem somewhat limited. We show ∼1.5% gain in unique alignments shown if mismatch tolerant aligner settings are used, although a more substantial difference could be seen when perfect matches were required. In practice, however, given a high-quality dataset like this, the benefits to a downstream application such as variant calling are likely to be small.

sensesThe second dataset, which had reads with substantially lower quality, illustrated that even reference-based tasks can benefit substantially from read preprocessing. Less than 25% of reads could be aligned by BWA without preprocessing. This could be improved to almost 80% by preprocessing, with almost 78% aligning even with strict settings.

unhoused_relationssensesIn the de novo assembly scenario, trimming was needed to ensure adapter sequences would not be incorporated into the newly assembled genome. This benefit was accompanied by a significant 58%/77% improvement in N50, respectively, and 28%/55% improvement, respectively, in maximum contig size for two datasets. The substantial improvement in assembly statistics further justifies the preprocessing of reads for de novo assembly.

limitationsIt is perhaps not surprising that preprocessing is so beneficial to de novo assembly, as many assembly tools, including velvet, do not exploit quality scores and thus treat all data equally, regardless of the known difference in quality. The effect of adapter sequences is also more serious, given the risk of incorporating adapter sequences into the final sequence assembly, compared with the mere reduction in the alignment rate typically seen in reference-based approaches.

5.2 Comparison against existing tools

concepts↗concept_edgesTrimmomatic compared favorably against all other tools in the tests performed.

When using high-quality raw data and liberal alignment criteria, the differences between the tools were relatively small. In this scenario, AdapterRemoval performed particularly well, reflecting its relative strength in removing technical sequences. This is unsurprising because, to the authors’ knowledge, AdapterRemoval is the only other tool to implement a pair-aware adapter removal strategy.

concepts↗concepts↗unhoused_relationscontent_tagsNonetheless, the use of strict alignment criteria, especially when combined with poor-quality input data, allows the differences between the tools to become clearer. In these scenarios, appropriate trimming based on quality seems to be more important, whereas technical sequence identification appeared to matter less. This helps explain the change in relative rankings of the tools between the two datasets. Trimmomatic with the Maximum Information mode seems to perform exceptionally well in these challenging scenarios.

Funding : We want to thank the BMBF for funding through grants 0315702F, 0315961 and 0315049A and BLE/BMELV Verbundprojekt: G 127/10 IF.

Conflict of Interest : none declared.

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claim_relations 4 · claim_type 1 · concept_edges 8 · concepts 67 · content_tags 48 · contribution 1 · controls 4 · limitations 10 · mechanisms 12 · method 1 · object_of_study 1 · phenomenon 1 · research_question 1 · senses 10 · unhoused_relations 16

Relations on this page carry a quotation for the claim. Where a relation was separately evidenced — and so tested against the vocabulary for its type — the row says so; 8 of 8 relations here carry that mark. The rest are the tagger’s reading of the sentence quoted beside them.

facettagfieldconf.whose words
object_of_studyTrimmomatic's paired-end-aware trimming algorithms, adaptive quality filtering, architecture, and effects on alignment and assemblyobject_of_studythe paper's words
research_questionhow to preprocess Illumina reads flexibly and efficiently while preserving pairing and improving downstream reference-based and de novo analysesresearch_questionthe paper's words
contributionTrimmomatic, a pair-aware pipeline with simple and palindrome adapter detection plus sliding-window and Maximum Information quality filteringcontributionthe paper's words
claim_typemethodclaim_typethe paper's words
phenomenontechnical-sequence contamination and position-dependent quality loss in Illumina sequence readsphenomenonthe paper's words
methodsystem-or-tool-buildingmethodthe paper's words
mechanismsA mismatched seed locates candidates, local alignment scores them, and the aligned region plus suffix is removed.mechanisms[0]0.95the tagger's words
mechanismsEqual valid length, reverse-complement sequence, and opposite-adapter contamination are tested together.mechanisms[1]0.95the tagger's words
mechanismsAdapters are prepended and the combined paired sequences are globally aligned.mechanisms[2]0.95the tagger's words
mechanismsSixteen bases become a 64-bit seed and XOR/popcount measures mismatches.mechanisms[3]0.95the tagger's words
mechanismsA moving mean-quality threshold removes the read suffix after consecutive poor bases.mechanisms[4]0.95the tagger's words
mechanismsTrimming becomes stricter later in a read as extra bases add less value and more error risk.mechanisms[5]0.95the tagger's words
mechanismsA logistic value curve rewards reads crossing a user-defined useful length.mechanisms[6]0.95the tagger's words
mechanismsRetained length contributes a linear information score.mechanisms[7]0.95the tagger's words
mechanismsPer-base correctness probabilities accumulate into a read error score.
also in: error factor
mechanisms[8]0.95the tagger's words
mechanismsThe peak of the length, coverage, and error score determines the trim point.mechanisms[9]0.95the tagger's words
mechanismsOrdered steps may inspect single reads or pairs while pairing state is tracked.mechanisms[10]0.95the tagger's words
mechanismsMultiple CPU cores improve preprocessing performance.mechanisms[11]0.95the tagger's words
limitationsShort partial adapters cannot satisfy simple mode's minimum overlap and are not detectable there.limitations[0]0.95the tagger's words
limitationsPalindrome mode works only on paired-end data and only detects adapter read-through.limitations[1]0.95the tagger's words
limitationsTrimmomatic's technical-sequence methods do not filter or classify barcode data.limitations[2]0.95the tagger's words
limitationsSequential single-purpose tools create large intermediate files and require pair awareness in every tool.limitations[3]0.95the tagger's words
limitationsA shell pipeline can lose read-pair relationships and cannot always assess the pair as a unit.limitations[4]0.95the tagger's words
limitationsLogging every read's trimming status has significant performance cost.limitations[5]0.95the tagger's words
limitationsSingle-end fallback makes alignments suboptimal in the cross-tool comparison.limitations[6]0.95the tagger's words
limitationsThe runtime benchmark cached a small dataset and may not represent I/O-limited practice.limitations[7]0.95the tagger's words
limitationsHigh-quality data with tolerant alignment showed only small preprocessing gains.limitations[8]0.95the tagger's words
limitationsAssemblers that ignore quality scores treat low- and high-quality bases equally.limitations[9]0.95the tagger's words
controlsuser-defined minimum overlap and alignment threshold
eliminates: false-positive simple-mode contaminant matches · outcome: held
controls[0]0.95the tagger's words
controlscombined read-through symptoms
eliminates: weak evidence from any one adapter symptom · outcome: held
controls[1]0.95the tagger's words
controlsalternative BWA aligner
eliminates: result dependence on Bowtie 2 alone · outcome: held
controls[2]0.95the tagger's words
controlsstrict and tolerant alignment settings
eliminates: conclusions tied to one mismatch tolerance · outcome: held
controls[3]0.95the tagger's words
conceptsnext-generation sequencingconcepts[0]0.95the paper's words
conceptsNGS
also in: NGS
concepts[1]0.95the paper's words
conceptsread preprocessing toolsconcepts[2]0.95the paper's words
conceptsTrimmomatic
also in: Trimmomatic
concepts[3]0.95the paper's words
conceptspaired-end dataconcepts[4]0.95the paper's words
conceptspoor quality
also in: poor quality
concepts[5]0.95the paper's words
conceptstechnical sequencesconcepts[6]0.95the paper's words
conceptssuboptimal downstream analysesconcepts[7]0.95the paper's words
conceptsFASTQ files
also in: FASTQ files
concepts[8]0.95the paper's words
conceptspositional relationshipconcepts[9]0.95the paper's words
conceptspair awarenessconcepts[10]0.95the paper's words
conceptssingletons
also in: singletons
concepts[11]0.95the paper's words
conceptsintermediate filesconcepts[12]0.95the paper's words
conceptsread trimming and filteringconcepts[13]0.95the paper's words
conceptsread preprocessingconcepts[14]0.95the paper's words
conceptssimple mode
also in: simple mode
concepts[15]0.95the paper's words
conceptsapproximate matchconcepts[16]0.95the paper's words
conceptsadapters
also in: adapters
concepts[17]0.95the paper's words
conceptsshort partial adapter sequencesconcepts[18]0.95the paper's words
conceptspolymerase chain reactionconcepts[19]0.95the paper's words
conceptsPCR
also in: PCR
concepts[20]0.95the paper's words
conceptspalindrome modeconcepts[21]0.95the paper's words
conceptsadapter read-throughconcepts[22]0.95the paper's words
conceptssensitivity and specificityconcepts[23]0.95the paper's words
conceptsseed and extendconcepts[24]0.95the paper's words
conceptslocal alignmentconcepts[25]0.95the paper's words
conceptsglobal alignmentconcepts[26]0.95the paper's words
concepts16-base fragmentconcepts[27]0.95the paper's words
concepts64-bit integerconcepts[28]0.95the paper's words
conceptsbitwise-XOR
also in: bitwise-XOR
concepts[29]0.95the paper's words
conceptspopcount
also in: popcount
concepts[30]0.95the paper's words
conceptsquality filtering alternativesconcepts[31]0.95the paper's words
conceptsIllumina quality scoreconcepts[32]0.95the paper's words
conceptsSliding Windowconcepts[33]0.95the paper's words
conceptssingle weak baseconcepts[34]0.95the paper's words
conceptsMaximum Informationconcepts[35]0.95the paper's words
conceptsadaptive approachconcepts[36]0.95the paper's words
conceptslength thresholdconcepts[37]0.95the paper's words
conceptscoverage
also in: coverage
concepts[38]0.95the paper's words
conceptserror rate
also in: error rate
concepts[39]0.95the paper's words
conceptscorrectness probabilitiesconcepts[40]0.95the paper's words
conceptspipeline-based architectureconcepts[41]0.95the paper's words
conceptspaired
also in: paired
concepts[42]0.95the paper's words
conceptssingle
also in: single
concepts[43]0.95the paper's words
conceptsmultiple threadsconcepts[44]0.95the paper's words
conceptsPerformance
also in: Performance
concepts[45]0.95the paper's words
conceptsphred+33
also in: phred+33
concepts[46]0.95the paper's words
conceptsphred+64
also in: phred+64
concepts[47]0.95the paper's words
conceptsreference-based alignmentconcepts[48]0.95the paper's words
conceptsBowtie 2
also in: Bowtie 2
concepts[49]0.95the paper's words
conceptsBWA
also in: BWA
concepts[50]0.95the paper's words
conceptsde novo assemblyconcepts[51]0.95the paper's words
conceptsVelvet
also in: Velvet
concepts[52]0.95the paper's words
conceptsuniquely aligned readsconcepts[53]0.95the paper's words
conceptsMaximum Information approachconcepts[54]0.95the paper's words
conceptsSliding Window approachconcepts[55]0.95the paper's words
conceptscontig N50 sizeconcepts[56]0.95the paper's words
conceptsmaximum contig sizeconcepts[57]0.95the paper's words
conceptsadapter sequenceconcepts[58]0.95the paper's words
conceptsAdapterRemovalconcepts[59]0.95the paper's words
conceptsScythe/Sickle
also in: Scythe/Sickle
concepts[60]0.95the paper's words
conceptsEA-Utils
also in: EA-Utils
concepts[61]0.95the paper's words
conceptsCutadapt
also in: Cutadapt
concepts[62]0.95the paper's words
conceptsexecution timeconcepts[63]0.95the paper's words
conceptsother tools
also in: other tools
concepts[64]0.95the paper's words
conceptsstrict alignment criteriaconcepts[65]0.95the paper's words
conceptspoor-quality input dataconcepts[66]0.95the paper's words
unhoused_relationsMaximum Information approach → Sliding Window approach
Maximum Information outperforms Sliding Window in both cases.
unhoused_relations[0]0.95the tagger's words
unhoused_relationsTrimmomatic → read preprocessing tools
Trimmomatic was developed as a flexible and efficient preprocessing tool.
unhoused_relations[1]0.95the tagger's words
unhoused_relationspair awareness → positional relationship
Pair awareness preserves the positional relationship between paired files.
unhoused_relations[2]0.95the tagger's words
unhoused_relationssimple mode → approximate match
Simple mode works by finding an approximate match to a technical sequence.
unhoused_relations[3]0.95the tagger's words
unhoused_relationssimple mode → short partial adapter sequences
Simple mode cannot detect very short partial adapter sequences.
unhoused_relations[4]0.95the tagger's words
unhoused_relationspalindrome mode → sensitivity and specificity
Palindrome mode has greater sensitivity and specificity than simple mode for read-through.
unhoused_relations[5]0.95the tagger's words
unhoused_relationssimple mode → local alignment
Simple mode uses local alignment.
unhoused_relations[6]0.95the tagger's words
unhoused_relationspalindrome mode → global alignment
Palindrome mode uses global alignment.
unhoused_relations[7]0.95the tagger's words
unhoused_relationspalindrome mode → adapter read-through
Palindrome mode is limited to detecting adapter read-through.
unhoused_relations[8]0.95the tagger's words
unhoused_relationsSliding Window → single weak base
Sliding-window averaging prevents one weak base from discarding later high-quality data.
unhoused_relations[9]0.95the tagger's words
unhoused_relationsMaximum Information → adaptive approach
Maximum Information implements the adaptive quality-filtering approach.
unhoused_relations[10]0.95the tagger's words
unhoused_relationspipeline-based architecture → paired
The pipeline tracks paired reads separately from single reads.
unhoused_relations[11]0.95the tagger's words
unhoused_relationsmultiple threads → Performance
Multiple threads improve performance when CPU cores are available.
unhoused_relations[12]0.95the tagger's words
unhoused_relationsread preprocessing → adapter sequence
Preprocessing prevents adapter sequence incorporation into de novo assemblies.
unhoused_relations[13]0.95the tagger's words
unhoused_relationsEA-Utils → singletons
EA-Utils maintains pairing but loses singleton reads.
unhoused_relations[14]0.95the tagger's words
unhoused_relationsstrict alignment criteria → other tools
Strict criteria and poor-quality data make performance differences among tools clearer.
unhoused_relations[15]0.95the tagger's words
sensesTrimmomatic as flexible, pair-aware Illumina preprocessingsenses[0]0.95the tagger's words
sensessimple mode as generic technical-sequence matching with short-overlap limitssenses[1]0.95the tagger's words
sensespalindrome mode as joint paired-read adapter-read-through detectionsenses[2]0.95the tagger's words
sensesseed as a 64-bit encoding of sixteen bases for fast mismatch countingsenses[3]0.95the tagger's words
sensesSliding Window as fixed local mean-quality suffix trimmingsenses[4]0.95the tagger's words
sensesMaximum Information as adaptive balance among read uniqueness, coverage, and errorsenses[5]0.95the tagger's words
sensespipeline architecture as user-ordered pair-aware processing stepssenses[6]0.95the tagger's words
sensespreprocessing value as conditional on input quality and downstream task stringencysenses[7]0.95the tagger's words
sensesreference-based benefit as improved alignment rates after contaminant and quality trimmingsenses[8]0.95the tagger's words
sensesde novo benefit as preventing adapter incorporation and improving assembly continuitysenses[9]0.95the tagger's words
claim_relationsbuilds-on → seed and extend alignment
note: Simple mode uses standard seed-and-extend alignment.
claim_relations[0]0.95the tagger's words
claim_relationsextends → adapter trimming
note: Palindrome mode adds pair-aware short read-through detection.
claim_relations[1]0.95the tagger's words
claim_relationscomplements → reference aligners
note: Preprocessing is evaluated before Bowtie 2 and BWA.
claim_relations[2]0.95the tagger's words
claim_relationscomplements → de novo assembly
note: Preprocessing improves Velvet assembly inputs.
claim_relations[3]0.95the tagger's words
concept_edgesnext-generation sequencing —same-as→ NGS
the relation itself is separately evidenced · strength of the relation: 1 · note: Parenthetical abbreviation equivalence. · the relation stated: Next-generation sequencing is abbreviated NGS.
concept_edges[0]0.95the tagger's words
concept_edgespolymerase chain reaction —same-as→ PCR
the relation itself is separately evidenced · strength of the relation: 1 · note: Parenthetical abbreviation equivalence. · the relation stated: Polymerase chain reaction is abbreviated PCR.
concept_edges[1]0.95the tagger's words
concept_edgespoor quality —causes→ suboptimal downstream analyses
direction on the measure: increases · the relation itself is separately evidenced · strength of the relation: 0.7 · note: Modal 'can' makes the causal strength partial. · the relation stated: Poor-quality sequence can result in suboptimal downstream analysis.
concept_edges[2]0.95the tagger's words
concept_edgestechnical sequences —causes→ suboptimal downstream analyses
direction on the measure: increases · the relation itself is separately evidenced · strength of the relation: 0.7 · note: Modal 'can' makes the causal strength partial. · the relation stated: Technical sequences can result in suboptimal downstream analysis.
concept_edges[3]0.95the tagger's words
concept_edgesread preprocessing —causes→ uniquely aligned reads
direction on the measure: increases · the relation itself is separately evidenced · strength of the relation: 0.95 · note: Explicit measured increase. · the relation stated: Preprocessing increased the number of uniquely aligned reads. Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but the span writes 'preprocessing', not 'read preprocessing'.
concept_edges[4]0.95the tagger's words
concept_edgesread preprocessing —causes→ contig N50 size
direction on the measure: increases · the relation itself is separately evidenced · strength of the relation: 1 · note: Direct measured increase. · the relation stated: Preprocessing increased contig N50 size by 58 percent. Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but the span writes 'preprocessing', not 'read preprocessing'.
concept_edges[5]0.95the tagger's words
concept_edgesread preprocessing —causes→ maximum contig size
direction on the measure: increases · the relation itself is separately evidenced · strength of the relation: 0.95 · note: Approximate measured increase. · the relation stated: Preprocessing improved maximum contig size by about 28 percent.
concept_edges[6]0.95the tagger's words
concept_edgesTrimmomatic —contrasted-with→ other tools
the relation itself is separately evidenced · strength of the relation: 0.95 · note: Direct comparison statement. · the relation stated: Trimmomatic compared favorably against all other tested tools.
concept_edges[7]0.95the tagger's words
content_tagsNo existing tool combination met the authors' flexibility, paired-end, and performance requirements.content_tags.findings[0]0.95the tagger's words
content_tagsTrimmomatic was at least competitive and often superior in every tested scenario.content_tags.findings[1]0.95the tagger's words
content_tagsSimple mode detects arbitrary technical sequences but misses short partial adapters.content_tags.findings[2]0.95the tagger's words
content_tagsPalindrome mode improves sensitivity and specificity for adapter read-through.content_tags.findings[3]0.95the tagger's words
content_tagsCombining both modes is required for comprehensive adapter detection.content_tags.findings[4]0.95the tagger's words
content_tagsSliding-window filtering avoids letting one weak base discard later high-quality sequence.content_tags.findings[5]0.95the tagger's words
content_tagsMaximum Information balances useful read length against accumulated error risk.content_tags.findings[6]0.95the tagger's words
content_tagsPreprocessing increased uniquely aligned reads, especially under strict alignment.content_tags.findings[7]0.95the tagger's words
content_tagsAfter trimming, almost 78% of low-quality dataset reads aligned perfectly.content_tags.findings[8]0.95the tagger's words
content_tagsPreprocessing increased first-dataset contig N50 by 58% and maximum contig length by about 28%.content_tags.findings[9]0.95the tagger's words
content_tagsSecond-dataset N50 improved about 77% and maximum contig length about 55%.content_tags.findings[10]0.95the tagger's words
content_tagsMaximum Information outperformed Sliding Window in both alignment cases.content_tags.findings[11]0.95the tagger's words
content_tagsTrimmomatic remained the best performer on the second dataset, especially in Maximum Information mode.content_tags.findings[12]0.95the tagger's words
content_tagsPreprocessing benefits were modest for high-quality mismatch-tolerant reference alignment but large for low-quality reads and de novo assembly.content_tags.findings[13]0.95the tagger's words
content_tagsStrict criteria and poor input quality made differences between tools clearer.content_tags.findings[14]0.95the tagger's words
content_tagsapproximate local matching of reads against user-supplied technical sequences
also in: simple mode
content_tags.definitions[0]0.95the tagger's words
content_tagspaired-end adapter-read-through detection using reverse-complement and adapter signalscontent_tags.definitions[1]0.95the tagger's words
content_tagsa fragment shorter than the read that leaves opposite-adapter sequence at read endscontent_tags.definitions[2]0.95the tagger's words
content_tagsinitial approximate seed matching followed by local or global alignmentcontent_tags.definitions[3]0.95the tagger's words
content_tags5-prime scanning that trims when mean window quality drops below thresholdcontent_tags.definitions[4]0.95the tagger's words
content_tagsadaptive choice of trim position from length threshold, coverage, and error factorscontent_tags.definitions[5]0.95the tagger's words
content_tagsminimum useful read length with a steep value transition around a targetcontent_tags.definitions[6]0.95the tagger's words
content_tagslinear information score based on retained sequence length
also in: coverage
content_tags.definitions[7]0.95the tagger's words
content_tagsaccumulated likelihood of errors derived from base-quality probabilities
also in: error rate
content_tags.definitions[8]0.95the tagger's words
content_tagsordered user-configurable steps operating on single reads or complete pairscontent_tags.definitions[9]0.95the tagger's words
content_tagsforward and reverse reads retaining their shared-fragment positional relationshipcontent_tags.definitions[10]0.95the tagger's words
content_tags16-base — fragment length used for initial seed comparisonscontent_tags.quantities[0]0.95the tagger's words
content_tags64-bit — integer width of encoded seedscontent_tags.quantities[1]0.95the tagger's words
content_tags0 to 1 — range of Maximum Information strictnesscontent_tags.quantities[2]0.95the tagger's words
content_tags2 × 100 bp — read geometry of dataset 1content_tags.quantities[3]0.95the tagger's words
content_tags2 × 250 bp — read geometry of dataset 2content_tags.quantities[4]0.95the tagger's words
content_tags<1.5% — raw reads aligned in perfect-match strict modecontent_tags.quantities[5]0.95the tagger's words
content_tagsalmost 78% — trimmed reads aligned perfectlycontent_tags.quantities[6]0.95the tagger's words
content_tags58% — first-dataset N50 gaincontent_tags.quantities[7]0.95the tagger's words
content_tags∼77% — second-dataset N50 gaincontent_tags.quantities[8]0.95the tagger's words
content_tags<20 000 reads — separation among top three tools under tolerant alignmentcontent_tags.quantities[9]0.95the tagger's words
content_tagsalignment scorecontent_tags.variables[0]0.95the tagger's words
content_tagsread quality
also in: read quality
content_tags.variables[1]0.95the tagger's words
content_tagsread length
also in: read length
content_tags.variables[2]0.95the tagger's words
content_tagslength thresholdcontent_tags.variables[3]0.95the tagger's words
content_tagscoverage
also in: coverage
content_tags.variables[4]0.95the tagger's words
content_tagserror rate
also in: error rate
content_tags.variables[5]0.95the tagger's words
content_tagsstrictness settingcontent_tags.variables[6]0.95the tagger's words
content_tagsquality stringency settingscontent_tags.variables[7]0.95the tagger's words
content_tagsuniquely aligned readscontent_tags.variables[8]0.95the tagger's words
content_tagscontig N50 sizecontent_tags.variables[9]0.95the tagger's words
content_tagsmaximum contig sizecontent_tags.variables[10]0.95the tagger's words
content_tagsexecution timecontent_tags.variables[11]0.95the tagger's words

Interpretive choices

Tags that could not be placed in the text:

What was deliberately not tagged, and why, is recorded per paper by the tagger. Where a paper has no such record, this page says so rather than implying that nothing was declined.

© 2026 Shir Sivroni · ORCID 0009-0000-2597-2824