Nucleic Acid Observatory Viral Metagenomics Pipeline

This Nextflow pipeline is designed to process metagenomic sequencing data, characterize overall taxonomic composition, and identify and quantify reads mapping to human-infecting viruses. It was developed as part of the Nucleic Acid Observatory project.

Pipeline description

Overview

The pipeline currently consists of two workflows, an index workflow and a run workflow. The former creates index and reference files that are used by the latter, and is intended to be run first, after which many instantiations of the latter workflow can use the same index output files.

The run workflow then consists of five phases:

1. A preprocessing phase, in which input files undergo adapter & quality trimming, deduplication, and ribodepletion.
2. A taxonomic profiling phase, in which Kraken2 is used to assess the overall taxonomic composition of the input data and assess how it changes across different steps in the preprocessing phase.
3. A viral identification phase, in which a custom multi-step pipeline based around Bowtie2 and Kraken2 is used to sensitively and specifically identify human-infecting virus (HV) reads in the input data for downstream analysis.
4. An optional BLAST validation phase, in which putative HV reads from phase 3 are checked against the NCBI nt database to evaluate the sensitivity and specificity of the HV identification process.
5. A final QC and output phase, in which FASTQC, MultiQC and other tools are used to assess the quality of the data produced by the pipeline at various steps and produce summarized output data for downstream analysis and visualization.

A slight simplification of the overall process is given by this mildly baffling diagram:

Index workflow

The index workflow is run by setting mode = "index" in the relevant config file, and is intended to be run once (per reasonable length of time) to generate static index and reference files which can then be used by many instantiations of the run workflow. Many of these index/reference files are derived from publicly available reference genomes or other resources, and should thus be updated and re-run periodically as new versions of these become available; however, to keep results comparable across datasets analyzed with the run workflow, this should be done relatively rarely.

Key inputs to the index workflow include:

• A URL linking to a suitable Kraken2 database for taxonomic profiling (typically the latest release of the k2_standard database).
• URLS for up-to-date releases of reference genomes for various common contaminant species that can confound the identification of HV reads (currently human, cow, pig, carp¹, mouse, E. coli).
• URLs to sequence databases for small and large ribosomal subunits from SILVA.
• Up-to-date links to VirusHostDB and NCBI Taxonomy.

Given these inputs, the index workflow:

• Obtains an up-to-date list of human-infecting viruses and their corresponding taxids from VirusHostDB, incorporates information about their taxonomic structure (in particular, their parent taxid) from NCBI, and generates a tab-separated database of human-infecting viral taxa.
• Generates a similar TSV for all viral taxa, including those that don't infect humans.
• Makes Bowtie2 indices from (1) all human-infecting viral genomes in Genbank², (2) the human genome, (3) common non-human contaminants, plus BBMap indices for (2) and (3).
• Downloads and extracts local copies of (1) the BLAST nt database, (2) the specified Kraken2 DB, (3) the SILVA rRNA reference files.

For more information, see workflows/index.nf and the associated subworkflows and modules.

Run workflow

Preprocessing phase

The run workflow begins by concatenating all libraries assigned to the same sample ID together³, then optionally subsampling the concatenated read files down to a specified number of reads⁴. The reads then undergo cleaning by Cutadapt, Trimmomatic, and FASTP, in that order; the first two of these only screen for adapter contamination, while the last both screens for adapters and trims low-quality and low-complexity sequences. Running three different adapter-trimming tools in succession is unusual, but necessary to minimize spurious identification of "human-viral" reads based on adapter contamination of the reference genomes.

Cleaned reads then undergo deduplication with Clumpify, a tool from the BBTools suite that identifies and collapses read pairs that are identical modulo some specified error rate. (Importantly, this deduplication phase does not remove reverse-complement duplicates.⁵) The deduplicated reads then undergo ribodepletion with BBDuk, which searches for ribosomal k-mers based on a SILVA sequencing database and removes any reads that exhibit rRNA matches above some threshold. This is performed twice in series, once with relatively strict parameters for what sequences qualify as "ribosomal" (which thus removes relatively fewer reads) and again with more inclusive parameters (which thus result in more reads being removed). The output of the initial, more conservative ribodepletion step is passed to the viral identification phase, while the output of the latter, more aggressive step is passed to the taxonomic profiling phase⁶.

Taxonomic profiling phase

In the taxonomic profiling phase, paired-end reads output from the preprocessing phase are merged together into single sequences through a combination of BBMerge (which aligns and merges read pairs with significant overlap) and end-to-end concatenation (with an intervening "N" base, for those read pairs BBMerge is unable to merge). The resulting single sequences are passed to Kraken2 for taxonomic assignment, using the reference database obtained in the index workflow. The output of Kraken2 is then passed to Bracken for correction and summarization, and the outputs of both Kraken and Bracken are merged across samples to produce single output files.

This phase is actually run three times across the pipeline: once on the cleaned reads, once on deduplicated reads, and once on the output of the second ribodepletion step. The latter of these is the primary source of data for overall assessment of the composition of the dataset; the former two are primarily used to assess how deduplication alters the measured taxonomic composition.

Viral identification phase

The goal of this phase is to sensitively and specifically identify reads arising from human-infecting virus reads. It uses a multi-step process designed to avoid false-positive and false-negative errors arising from simpler systems tried previously.

1. To begin with, the output of the initial (conservative) ribodepletion step is aligned against a database of human-infecting viral genomes generated from Genbank by the index workflow. This initial alignment is conducted with Bowtie2 using quite permissive parameters designed to capture as many putative HV reads as possible. The SAM and FASTQ files are processed to generate new read files containing any read pair for which at least one read matches the HV database.
2. The output of the previous step is passed to a filtering step, in which reads matching a series of common contaminant sequences are removed. This is done by aligning surviving reads to these contaminants using both Bowtie2 and BBMap in series⁷. Contaminants to be screened against include reference genomes from human, cow, pig, carp, mouse and E. coli, as well as various genetic engineering vectors.
3. Following filtering, surviving read pairs are merged into single sequences as described for the taxonomic profiling phase, then undergo an additional deduplication step intended to remove reverse-complement duplicates that weren't detected during preprocessing.
4. After deduplication, surviving merged reads are passed to Kraken2 (using the same reference database used in the taxonomic profiling phase). For each read, we record whether that read was (1) assigned to a human-infecting virus taxon with Kraken, (2) assigned to a non-HV taxon with Kraken, or (3) not assigned to any taxon. All reads in category (2) are filtered out.
5. Finally, reads are assigned a final HV status if they:
   • Are given matching HV assignments by both Bowtie2 and Kraken2; or
   • Are unassigned by Kraken and align to an HV taxon with Bowtie2 with an alignment score above a user-specifed threshold⁸.

Following HV status assignment, information for each read pair is processed into a TSV file available in the output directory as hv_hits_putative_collapsed.tsv.gz. Finally, the number of read pairs mapping to each detected HV taxon is counted and output as hv_clade_counts.tsv.gz for downstream use.

BLAST validation phase

To evaluate the performance of the process described in the viral identification phase, it's useful to get some ground-truth information about whether the human-viral assignments made in that subworkflow are correct. To do this, we use BLASTN to align the putative HV reads output by the previous phase against the nt database, then process the output to check whether each sequence had a high-scoring alignment to at least one HV sequence. For computational tractability, this can be performed on only a subset of surviving HV reads (specified by params.blast_hv_fraction)⁹.

Currently, this subworkflow performs the BLAST alignment, filters & summarizes the output to a manageable size, and saves the result to the output directory as blast_hits_paired.tsv.gz. Actual assessment of HV status and performance evaluation is currently performed external to this workflow.

QC and output phase

Each step in the preprocessing phase generates paired FASTQ files that can be analyzed with FASTQC and MultiQC. Doing so allows us to track important metrics (e.g. read quality, read numbers, adapter content) across the pipeline. This phase takes the MultiQC outputs from each phase and extracts relevant metrics into easy-to-parse TSV files¹⁰ for downstream processing.

In addition, for each iteration of the taxonomic classification workflow, this phase takes the Bracken output data and combines it with MultiQC metrics to compute the number and fraction of input reads assigned to each of the following categories (if applicable):

• Filtered (removed during cleaning)
• Duplicate (removed during deduplication)
• Ribosomal (removed during ribodepletion)
• Unassigned (non-ribosomal reads that were not assigned to any taxon by Kraken/Bracken)
• Bacterial (non-ribosomal reads assigned to the Bacteria domain by Kraken/Bracken)
• Archaeal (non-ribosomal reads assigned to the Archaea domain by Kraken/Bracken)
• Viral (non-ribosomal reads assigned to the Viruses domain by Kraken/Bracken)
• Human (non-ribosomal reads assigned to the Eukarya domain¹¹ by Kraken/Bracken)

This composition information is given in a file named taxonomic_composition.tsv.gz in the results directory.

Pipeline outputs

If the pipeline runs to completion, the following output files are expected.

Index workflow

1. output/input/index-params.json: JSON file giving all the parameters passed to the pipeline (useful for trying to reproduce someone else's results).
2. output/input/pipeline-version.txt: Version of the pipeline with which index directory was created.
3. output/input/time.txt: Start time of index workflow run.
4. output/results/nt: Directory containing extracted BLAST database files for BLASTing against nt.
5. output/results/bt2-hv-index: Directory containing Bowtie2 index for human-infecting viral genomes.
6. output/results/bt2-human-index: Directory containing Bowtie2 index for the human genome.
7. output/results/bt2-other-index: Directory containing Bowtie2 index for other contaminant sequences.
8. output/results/bbm-human-index: Directory containing BBMap index for the human genome.
9. output/results/bbm-other-index: Directory containing BBMap index for other contaminant sequences.
10. output/results/kraken_db: Directory containing Kraken2 reference database (by default, the most recent version of Standard).
11. output/results/human-viral-genomes-filtered.fasta.gz: FASTA file containing human-viral genomes downloaded from viral Genbank (filtered to remove transgenic, contaminated, or erroneous sequences).
12. output/results/genomeid-to-taxid.json: JSON mapping between HV taxids and NCBI genome IDs for the sequences in (8).
13. output/results/ribo-ref-concat.fasta.gz: Reference database of ribosomal LSU and SSU sequences from SILVA.
14. output/results/taxonomy-nodes.dmp: Taxonomy dump file from NCBI mapping between taxids and their parents in the NCBI taxonomy tree structure.
15. output/results/taxonomy-names.dmp: Taxonomy dump file from NCBI mapping between taxids and taxon names.
16. output/results/human-virus-db.tsv.gz: Database generated from (8), (9), (12) and (13) giving, for each human-viral taxon:
    • The taxid (taxid)
    • The taxid of the parent taxon (parent_taxid)
    • The scientific name (name)
    • The taxonomic rank (rank)
17. output/results/total-virus-db.tsv.gz: As (14), but for all viral taxa (including non-human-infecting ones).

Run workflow

1. output/input: Directory containing saved input information (useful for trying to reproduce someone else's results)
   1. adapters.fasta: FASTA file of adapter sequences used for adapter screening.
   2. params.json: JSON file giving all the parameters passed to the pipeline.
   3. A CSV file giving sample metadata (filename specified by params.sample_tab).
   4. time.txt: Start time of run workflow.
   5. pipeline-version.txt: Version of pipeline used for run.
   6. pipeline-version-index.txt: Version of pipeline used to generate index directory (params.ref_dir).
   7. index-params.json: JSON file giving parameters used to generate index directory (params.ref_dir).
2. output/intermediates: Intermediate files produced by key stages in the run workflow, saved for nonstandard downstream analysis.
   1. reads/cleaned: Directory containing paired FASTQ files for cleaned reads (i.e. the output of the preprocessing phase described above).
3. output/results: Directory containing processed results files for standard downstream analysis.
   1. hv/hv_hits_putative_collapsed.tsv.gz: TSV output by the viral identification phase, giving information about each read pair assigned to a human-infecting virus.
   2. hv/hv_clade_counts.tsv.gz: Summary of the previous file giving the number of HV read pairs mapped to each viral taxon. Includes both read pairs mapped directly to that taxon (n_reads_direct) and to that taxon plus all descendent taxa (n_reads_clade).
   3. hv/blast_hits_paired.tsv.gz: Summarized BLASTN output for putative HV read pairs, giving, for each read pair and subject taxid:
      • The number of reads in the read pair with high-scoring matches to that taxid (n_reads).
      • The best bitscores of alignments to that taxid for each matching read (bitscore_max and bitscore_min)¹².
   4. qc/qc_basic_stats.tsv.gz: Summary statistics for each sample at each stage of the preprocessing phase (stage), including:
      • GC content (percent GC);
      • Average read length (mean_seq_len);
      • Number of read pairs (n_read pairs);
      • Approximate number of base pairs in reads (n_bases_approx);
      • Percent duplicates as measured by FASTQC (percent_duplicates);
      • Pass/fail scores for each test conducted by FASTQC.
   5. qc/qc_adapter_stats.tsv.gz: Adapter statistics calculated by FASTQC for each sample and preprocessing stage, given as a percentage of reads containing adapter content (pc_adapters) at each position along the read (position) for each adapter detected (adapter) for each read in the read pair (read_pair).
   6. qc/qc_quality_base_stats.tsv.gz: Per-base read-quality statistics calculated by FASTQC for each sample and preprocessing stage, given as the mean Phred score (mean_phred_score) at each position along the read (position) for each read in the read pair (read_pair).
   7. qc/qc_quality_sequence_stats.tsv.gz: Per-sequence read-quality statistics calculated by FASTQC for each sample and preprocessing stage, given as the number of reads (n_sequences) with a given mean Phred score (mean_phred_score) for each read in the read pair (read_pair).
   8. taxonomy_final/taxonomic_composition.tsv.gz: High-level classification of input reads for each sample by preprocessing stage and taxonomic domain, based on taxonomic analysis of the complete workflow.
   9. taxonomy_final/kraken_reports.tsv.gz: Kraken output reports in TSV format, labeled by sample, based on taxonomic analysis of the complete workflow.
   10. taxonomy_pre_dedup/taxonomic_composition.tsv.gz: High-level classification of input reads for each sample by preprocessing stage and taxonomic domain, based on taxonomic analysis of the cleaned reads (i.e. without deduplication or ribodepletion).
   11. taxonomy_pre_dedup/kraken_reports.tsv.gz: Kraken output reports in TSV format, labeled by sample, based on taxonomic analysis of the cleaned reads (i.e. without deduplication or ribodepletion).
   12. taxonomy_post_dedup/taxonomic_composition.tsv.gz: High-level classification of input reads for each sample by preprocessing stage and taxonomic domain, based on taxonomic analysis of the deduplicated reads (i.e. without ribodepletion).
   13. taxonomy_post_dedup/kraken_reports.tsv.gz: Kraken output reports in TSV format, labeled by sample, based on taxonomic analysis of the deduplicated reads (i.e. without ribodepletion).

Using the workflow

Profiles and modes

The pipeline can be run in two modes, "index" and "run", each of which calls the corresponding workflow from the workflows directory.

For both modes, the pipeline can be run in multiple ways by modifying various configuration variables specified in configs/profiles.config. Currently, three profiles are implemented, all of which assume the workflow is being launched from an AWS EC2 instance:

• The batch profile is the default and attempts to run the pipeline with AWS Batch. This is the quickest and most efficient way to run the pipeline, but requires significant additional setup not described in this repo. To set up AWS Batch for this pipeline, follow the instructions here (steps 1-3), then modify your config file to point process.queue to the name of your Batch job queue.
• The ec2_local profile attempts to run the whole workflow locally on your EC2 instance, storing intermediate and outflow files on instance-linked block storage. This is simple and can be relatively fast, but is bottlenecked by your instance's CPU and memory allocations; in particular, if you don't use an instance with very high memory, the pipeline is likely to fail when loading the Kraken2 reference DB.
• The ec2_s3 profile runs the pipeline on your EC2 instance, but attempts to read and write files to a specified S3 directory. This avoids problems caused by insufficient storage on your EC2 instance, but (1) is significantly slower and (2) is still constrained by your instance's memory allocation.

To run the pipeline with a specified profile, run nextflow run PATH_TO_REPO_DIR -profile PROFILE_NAME -resume. Calling the pipeline without specifying a profile will run the batch profile by default. Future example commands in this README will assume you are using Batch; if you want to instead use a different profile, you'll need to modify the commands accordingly.

Tip

It's highly recommended that you always run nextflow run with the -resume option enabled. It doesn't do any harm if you haven't run a workflow before, and getting into the habit will help you avoid much sadness when you want to resume it without rerunning all your jobs.

Installation & setup

1. Install dependencies

To run this workflow with full functionality, you need access to the following dependencies:

1. SDKMAN!: To install the SDKMAN! Java SDK manager, follow the installation instructions available here.
2. Nextflow: To install the workflow management framework, follow the installation instructions available here, beginning by installing a recommended Java distribution through SDKMAN!.
3. Docker: To install Docker Engine for command-line use, follow the installation instructions available here (or here for installation on an AWS EC2 instance).
4. AWS CLI: If not already installed, install the AWS CLI by following the instructions available here.
5. Git: To install the Git version control tool, follow the installation instructions available here.

2. Configure AWS & Docker

To run the workflow using AWS S3 for the working and output directories, you first need to configure AWS access. To do this, you need to create a file at ~/.aws/config or ~/.aws/credentials specifying your access key ID and secret access key, e.g.

~/.aws/config:

[default]
region = us-east-1
output = table
tcp_keepalive = true
aws_access_key_id = <ACCESS_KEY_ID>
aws_secret_access_key = <SECRET_ACCESS_KEY>

~/.aws/credentials:

[default]
aws_access_key_id = <ACCESS_KEY_ID>
aws_secret_access_key = <SECRET_ACCESS_KEY>

If you encounter AccessDenied errors after doing this, you may also need to export these keys as environment variables before running Nextflow:

eval "$(aws configure export-credentials --format env)"

Next, you need to make sure your user is configured to use Docker. To do this, create the docker user group and add your current user to it:

sudo groupadd docker
sudo usermod -aG docker $USER
newgrp docker
docker run hello-world

3. Clone this repository

Clone this repo into a new directory as normal.

4. Run index/reference workflow

Tip

If someone else in your organization already uses this pipeline, it's likely they've already run the index workflow and generated an output directory. If this is the case, you can reduce costs and increase reproducibility by using theirs instead of generating your own. If you want to do this, skip this step, and edit configs/run.config such that params.ref_dir points to INDEX_DIR/output/results.

Create a new directory outside the repo directory and copy over the index workflow config file as nextflow.config in that directory:

mkdir index
cd index
cp REPO_DIR/configs/index.config nextflow.config

Next, edit nextflow.config such that params.base_dir points to the directory (likely on S3) where you want to store your index files. (You shouldn't need to modify anything else about the config file at this stage.)

Next, call nextflow run pointing at the repo directory (NB: not main.nf or any other workflow file; pointing to the directory will cause Nextflow to automatically run main.nf from that directory.):

nextflow run PATH_TO_REPO_DIR -resume

Wait for the workflow to run to completion; this is likely to take several hours at least.

Testing & validation

To confirm that the pipeline works in your hands, we provide a small test dataset (test/raw) to run through the run workflow. This can be used to test any of the pipeline profiles described above.

If your EC2 instance has the resources to handle it, the simplest way to start using the pipeline is to run the test data through it locally on that instance (i.e. without using S3). To do this:

1. Navigate to the test directory.
2. Edit nextflow.config to set params.ref_dir to the index directory you chose or created above (specifically PATH_TO_REF_DIR/output/results).
3. Still within the test directory, run nextflow run -profile ec2_local .. -resume.
4. Wait for the workflow to finish. Inspect the output directory to view the processed output files.

If this is successful, the next level of complexity is to run the workflow with a working directory on S3. To do this:

1. Within the test directory, edit nextflow.config to set params.base_dir to the S3 directory of your choice.
2. Still within that directory, run nextflow run -profile ec2_s3 .. -resume.
3. Wait for the workflow to finish, and inspect the output on S3.

Finally, you can run the test dataset through the pipeline on AWS Batch. To do this, configure Batch as described here (steps 1-3), then:

1. Within the test directory, edit nextflow.config to set params.base_dir to a different S3 directory of your choice and process.queue to the name of your Batch job queue.
2. Still within that directory, run nextflow run -profile batch .. -resume (or simply nextflow run .. -resume).
3. Wait for the workflow to finish, and inspect the output on S3.

Running on new data

To run the workflow on another dataset, you need:

1. Accessible raw data files in Gzipped FASTQ format, named appropriately.
2. A library file specifying the relationship between the names of these files (specifically, substrings uniquely identifying each pair of FASTQ files) and the original samples.
3. A sample sheet giving metadata for each sample (can be identical with the library file).
4. A config file in a clean launch directory, pointing to:
   • The directory containing the raw data (params.raw_dir).
   • The base directory in which to put the working and output directories (params.base_dir).
   • The directory containing the outputs of the reference workflow (params.ref_dir).
   • The library file (params.library_tab) and sample sheet (params.sample_tab).
   • Various other parameter values.

Note

Currently, the pipeline requires the following of raw data files:

• They must be contained in a single directory
• Each pair of reads files must have names ending in _1.fastq.gz and _2.fastq.gz, respectively
• Each pair of read files must be uniquely identifiable by a filename substring (specified in the library column of params.library_tab)

Note

The library file should specify the mapping between library read files and sample IDs. It must be a CSV file with library and sample columns, as well as any other metadata columns you feel is appropriate. See test/libraries.csv for a minimal example.

If running on Batch, a good process for starting the pipeline on a new dataset is as follows:

1. Process the raw data to have appropriate filenames (see above) and deposit it in an accessible S3 directory.
2. Create a clean launch directory and copy configs/run.config to a file named nextflow.config in that directory.
3. Create a library metadata file in that launch directory, specifying library/sample mappings and any other metadata (see above).
4. Edit nextflow.config to specify each item in params as appropriate, as well as setting process.queue to the appropriate Batch queue.
5. Run nextflow run PATH_TO_REPO_DIR -resume.
6. Navigate to {params.base_dir}/output to view and download output files.

Troubleshooting

When attempting to run a released version of the pipeline, the most common sources of errors are AWS permission issues. Before debugging a persistent error in-depth, make sure that you have all the permissions specified in Step 0 of our Batch workflow guide. Next, make sure Nextflow has access to your AWS credentials, such as by running eval "$(aws configure export-credentials --format env)".

Another common issue is for processes to fail with some variation of the following Docker-related error:

docker: failed to register layer: write /usr/lib/jvm/java-11-openjdk-amd64/lib/modules: **no space left on device**.

This is a fairly well-known problem, which can arise even when there is substantial free storage space accessible to your EC2 instance. Following the steps recommended here or here typically resolves the issue, either by deleting Docker assets to free up space (e.g. via docker system prune --all --force) or by giving Docker more space.

We will add more common failure modes here as they get reported.

Footnotes

1. The carp genome is included as a last-ditch attempt to capture any remaining Illumina adapter sequences before moving on to HV identification. I'm not especially confident this is helpful. ↩

2. Excluding transgenic, contaminated, or erroneous sequences, which are excluded according to a list of sequence ID patterns specified in the config file. ↩

3. This is controlled by the library file specied in the config file; any libraries with the same entry in the sample column are concatenated. This is primarily useful in cases where the same library is sequenced multiple times, e.g. to reach some total target depth. ↩

4. This is mainly useful for an initial run of the pipeline to confirm everything functions well for a given dataset and sample sheet; it's not recommended for analyses that are actually expected to yield meaningful results. ↩

5. Unfortunately, I'm not aware of any deduplication tool that (1) works well on paired-read data, (2) can detect and remove duplicates in the presence of sequencing errors, and (3) can handle reverse-complement duplicates. If you know of one, please do let me know! ↩

6. The splitting of ribodepletion into these two phases arises from differences in the needs of the two downstream processes they feed into. For viral identification, we want to make sure we aren't losing any HV reads through spurious identification as ribosomal, so we use more conservative parameters. For taxonomic profiling, we're much less concerned about the status of individual reads and more concerned about accurately measuring ribosomal content, so a more aggressive approach is appropriate. ↩

7. We've found in past investigations that the two aligners detect different contaminant sequences, and aligning against both is more effective at avoiding false positives than either in isolation. ↩

8. Specifically, Kraken-unassigned read pairs are classed as human-viral if, for either read in the pair, $S/ln(L) >= T$, where $S$ is the best-match Bowtie2 alignment score for that read, $L$ is the length of the read, and $T$ is the value of params.bt2_score_threshold specified in the config file. ↩

9. Setting params.blast_hv_fraction to 0 skips this step altogether. ↩

10. Specifically, qc_basic_stats.tsv.gz, qc_adapter_stats.tsv.gz, qc_quality_base_stats.tsv.gz and qc_quality_sequence_stats.tsv.gz. ↩

11. As human is the only eukaryotic genome included in the Standard reference database for Kraken2, all sequences assigned to that domain can be assigned to Homo sapiens. ↩

12. If only one read aligns to the target, these two fields will be identical. If not, they will give the higher and lower of the best bitscores for the two reads in the pair.. ↩