MUSCLE: a multiple sequence alignment method with reduced time and space complexity

BACKGROUND: In a previous paper, we introduced MUSCLE, a new program for creating multiple alignments of protein sequences, giving a brief summary of the algorithm and showing MUSCLE to achieve the highest scores reported to date on four alignment accuracy benchmarks. Here we present a more complete discussion of the algorithm, describing several previously unpublished techniques that improve biological accuracy and / or computational complexity. We introduce a new option, MUSCLE-fast, designed for high-throughput applications. We also describe a new protocol for evaluating objective functions that align two profiles. RESULTS: We compare the speed and accuracy of MUSCLE with CLUSTALW, Progressive POA and the MAFFT script FFTNS1, the fastest previously published program known to the author. Accuracy is measured using four benchmarks: BAliBASE, PREFAB, SABmark and SMART. We test three variants that offer highest accuracy (MUSCLE with default settings), highest speed (MUSCLE-fast), and a carefully chosen compromise between the two (MUSCLE-prog). We find MUSCLE-fast to be the fastest algorithm on all test sets, achieving average alignment accuracy similar to CLUSTALW in times that are typically two to three orders of magnitude less. MUSCLE-fast is able to align 1,000 sequences of average length 282 in 21 seconds on a current desktop computer. CONCLUSIONS: MUSCLE offers a range of options that provide improved speed and / or alignment accuracy compared with currently available programs. MUSCLE is freely available at

Accuracy of microbial community diversity estimated by closed- and open-reference OTUs

Next-generation sequencing of 16S ribosomal RNA is widely used to survey microbial communities. Sequences are typically assigned to Operational Taxonomic Units (OTUs). Closed- and open-reference OTU assignment matches reads to a reference database at 97% identity (closed), then clusters unmatched reads using a de novo method (open). Implementations of these methods in the QIIME package were tested on several mock community datasets with 20 strains using different sequencing technologies and primers. Richness (number of reported OTUs) was often greatly exaggerated, with hundreds or thousands of OTUs generated on Illumina datasets. Between-sample diversity was also found to be highly exaggerated in many cases, with weighted Jaccard distances between identical mock samples often close to one, indicating very low similarity. Non-overlapping hyper-variable regions in 70% of species were assigned to different OTUs. On mock communities with Illumina V4 reads, 56% to 88% of predicted genus names were false positives. Biological inferences obtained using these methods are therefore not reliable

Inhibitory Plasticity in a Lateral Band Improves Cortical Detection of Natural Vocalizations

SummaryThe interplay between excitation and inhibition in the auditory cortex is crucial for the processing of acoustic stimuli. However, the precise role that inhibition plays in the distributed cortical encoding of natural vocalizations has not been well studied. We recorded single units (SUs) and local field potentials (LFPs) in the awake mouse auditory cortex while presenting pup isolation calls to animals that either do (mothers) or do not (virgins) recognize the sounds as behaviorally relevant. In both groups, we observed substantial call-evoked inhibition. However, in mothers this was earlier, longer, stronger, and more stereotyped compared to virgins. This difference was most apparent for recording sites tuned to tone frequencies lower than the pup calls' high-ultrasonic frequency range. We hypothesize that this auditory cortical inhibitory plasticity improves pup call detection in a relatively specific manner by increasing the contrast between call-evoked responses arising from high-ultrasonic and lateral frequency neural populations

Effect of Wean-to-Finish Management on Pig Performance

An experiment consisting of three trials was conducted to determine the effect of wean-to-finish management systems on pig performance. Treatments consisted of: 1) wean-to-finish single stock (WF) at 7.5 ft2/pig from weaning (17 day mean age) to slaughter in a fully slatted finishing facility; 2) double stock (DS) at 3.75 ft2/pig for eight weeks following weaning and then split into two pens at 7.5 ft2/pig each; and 3) nursery (NF) at 3.75 ft2/ pig for eight weeks in a conventional nursery followed by movement to the finisher and stocked at 7.5 ft2/pig to slaughter. All pens had one two-hole wean-finish dry feeder per 15 pigs and one cup-drinker per 15 pigs. While there were health related performance problems in Trials 1 and 2 due to PRRS, there were no trial by treatment interactions. At the end of eight weeks, WF pigs were heavier (P\u3c.01) than DS pigs with NF pigs intermediate in weight (63.1, 59.2, and 60.9 lbs, respectively). The heavier weight was due to a difference (P\u3c.01) in feed intake between the WF and DS treatments. There was no effect of nursery phase treatment on feed efficiency. There was no effect (P\u3e.1) of any management treatment on any grow-finish phase production parameter reported. These data suggest that the performance improvement associated with wean–to-finish production systems occurs during the first eight weeks post-weaning. They also suggest that the response can be expected even when health challenges occur in a production system

Effect of Wean-to-Finish Management on Pig Performance

An experiment consisting of three trials was conducted to determine the effect of wean-to-finish management systems on pig performance. Treatments consisted of: 1) wean-to-finish single stock (WF) at 7.5 ft2/pig from weaning (17 day mean age) to slaughter in a fully slatted finishing facility; 2) double stock (DS) at 3.75 ft2/pig for eight weeks following weaning and then split into two pens at 7.5 ft2/pig each; and 3) nursery (NF) at 3.75 ft2/ pig for eight weeks in a conventional nursery followed by movement to the finisher and stocked at 7.5 ft2/pig to slaughter. All pens had one two-hole wean-finish dry feeder per 15 pigs and one cup-drinker per 15 pigs. While there were health related performance problems in Trials 1 and 2 due to PRRS, there were no trial by treatment interactions. At the end of eight weeks, WF pigs were heavier (P\u3c.01) than DS pigs with NF pigs intermediate in weight (63.1, 59.2, and 60.9 lbs, respectively). The heavier weight was due to a difference (P\u3c.01) in feed intake between the WF and DS treatments. There was no effect of nursery phase treatment on feed efficiency. There was no effect (P\u3e.1) of any management treatment on any grow-finish phase production parameter reported. These data suggest that the performance improvement associated with wean–to-finish production systems occurs during the first eight weeks post-weaning. They also suggest that the response can be expected even when health challenges occur in a production system

UCHIME improves sensitivity and speed of chimera detection

Motivation: Chimeric DNA sequences often form during polymerase chain reaction amplification, especially when sequencing single regions (e.g. 16S rRNA or fungal Internal Transcribed Spacer) to assess diversity or compare populations. Undetected chimeras may be misinterpreted as novel species, causing inflated estimates of diversity and spurious inferences of differences between populations. Detection and removal of chimeras is therefore of critical importance in such experiments

PILER-CR: Fast and accurate identification of CRISPR repeats

BACKGROUND: Sequencing of prokaryotic genomes has recently revealed the presence of CRISPR elements: short, highly conserved repeats separated by unique sequences of similar length. The distinctive sequence signature of CRISPR repeats can be found using general-purpose repeat- or pattern-finding software tools. However, the output of such tools is not always ideal for studying these repeats, and significant effort is sometimes needed to build additional tools and perform manual analysis of the output. RESULTS: We present PILER-CR, a program specifically designed for the identification and analysis of CRISPR repeats. The program executes rapidly, completing a 5 Mb genome in around 5 seconds on a current desktop computer. We validate the algorithm by manual curation and by comparison with published surveys of these repeats, finding that PILER-CR has both high sensitivity and high specificity. We also present a catalogue of putative CRISPR repeats identified in a comprehensive analysis of 346 prokaryotic genomes. CONCLUSION: PILER-CR is a useful tool for rapid identification and classification of CRISPR repeats. The software is donated to the public domain. Source code and a Linux binary are freely available at