Basic Sequence QC

A generic pipeline that can be run on an arbitrary set of Illumina sequence files, regardless of the project or organism of interest.

• Sequence quality information

Analyses

• fastp: Collect sequence QC stats
• hostile: Removal of host (human) reads (optional)

Usage

nextflow run BCCDC-PHL/basic-sequence-qc \
  [--prefix 'prefix'] \
  --fastq_input <your fastq input directory> \
  --outdir <output directory>

Alternatively, a sample_sheet.csv file can be provided, with fields:

ID
R1
R2

For example:

ID,R1,R2
SAMPLE-01,/path/to/SAMPLE-01_R1.fastq.gz,/path/to/SAMPLE-01_R2.fastq.gz
SAMPLE-02,/path/to/SAMPLE-02_R1.fastq.gz,/path/to/SAMPLE-02_R2.fastq.gz
SAMPLE-03,/path/to/SAMPLE-03_R1.fastq.gz,/path/to/SAMPLE-03_R2.fastq.gz

The sample_sheet.csv file can be provided using the --sample_sheet_input flag as follows:

nextflow run BCCDC-PHL/basic-sequence-qc \
  [--prefix 'prefix'] \
  --sample_sheet_input <your sample_sheet.csv file> \
  --outdir <output directory>

Publishing Trimmed Reads

By default, the trimmed reads are not 'published' to the output directory. To enable publishing the trimmed reads, add the --publish_trimmed_reads flag:

nextflow run BCCDC-PHL/basic-sequence-qc \
  [--prefix 'prefix'] \
  --publish_trimmed_reads \
  --sample_sheet_input <your sample_sheet.csv file> \
  --outdir <output directory>

Dehosting

This pipeline supports an optional dehosting step that can be used to remove human-derived sequence reads. Removal of host reads from other organisms is not currently supported.

Details on available references can be found on the bede/hostile README.

Setup

This pipeline assumes that the host genome index used for dehosting has already been downloaded to the directory associated with the hostile_index_dir parameter.

The list of available reference names can be found by running:

hostile index list

...which should return a list like this:

Remote  human-t2t-hla
Remote  human-t2t-hla-argos985
Remote  human-t2t-hla-argos985-mycob140
Remote  human-t2t-hla.rs-viral-202401_ml-phage-202401
Remote  human-t2t-hla.argos-bacteria-985_rs-viral-202401_ml-phage-202401
Remote  mouse-mm39
Local   human-t2t-hla (Bowtie2)
Local   human-t2t-hla-argos985-mycob140 (Minimap2, Bowtie2)

Indexes can be downloaded using the following command:

hostile index fetch -n <index_name>

Performing Dehosting

To include the dehosting step in the pipeline analysis, add the --dehost flag:

nextflow run BCCDC-PHL/basic-sequence-qc \
  -profile conda \
  --cache ~/.conda/envs \
  --fastq_input /path/to/fastq_input \
  --dehost \
  --outdir /path/to/output-dir

By default, the human-t2t-hla reference will be used. To use an alternative reference, include the --dehosting_index flag along with the reference name:

nextflow run BCCDC-PHL/basic-sequence-qc \
  -profile conda \
  --cache ~/.conda/envs \
  --fastq_input /path/to/fastq_input \
  --dehost \
  --dehosting_index human-t2t-hla-argos985-mycob140 \
  --outdir /path/to/output-dir

Output

The basic output directory structure is:

outdir
├── basic_qc_stats.csv
├── sample-01
│   ├── sample-01_fastp.csv
│   ├── sample-01_fastp.json
│   └── sample-01_fastp.html
├── sample-02
│   ├── sample-02_fastp.csv
│   ├── sample-02_fastp.json
│   └── sample-02_fastp.html
├── sample-03
│   ├── ...
...

A single output file in .csv format will be created in the directory specified by --outdir. The filename will be basic_qc_stats.csv. If a prefix is provided using the --prefix flag, it will be prepended to the output filename, for example: prefix_basic_qc_stats.csv.

The output file includes the following headers:

sample_id
total_reads_before_filtering
total_reads_after_filtering
total_bases_before_filtering
total_bases_after_filtering
read1_mean_length_before_filtering
read1_mean_length_after_filtering
read2_mean_length_before_filtering
read2_mean_length_after_filtering
q20_bases_before_filtering
q20_bases_after_filtering
q20_rate_before_filtering
q20_rate_after_filtering
q30_bases_before_filtering
q30_bases_after_filtering
q30_rate_before_filtering
q30_rate_after_filtering
gc_content_before_filtering
gc_content_after_filtering
adapter_trimmed_reads
adapter_trimmed_bases
read1_num_poly_g_before_filtering
read1_num_poly_g_after_filtering
read2_num_poly_g_before_filtering
read2_num_poly_g_after_filtering
duplication_rate

For each sample, a separate output dir will be created below the --outdir, named using the sample ID and containing csv, json and html-formatted fastp reports for that sample.

Trimmed Reads

If the --publish_trimmed_reads flag is included, the trimmed reads will be included in the output. If no dehosting is performed, these files will be named:

<SAMPLE_ID>/<SAMPLE_ID>_trimmed_R1.fastq.gz
<SAMPLE_ID>/<SAMPLE_ID>_trimmed_R2.fastq.gz

If dehosting is performed, the trimmed reads will be named:

<SAMPLE_ID>/<SAMPLE_ID>_pre-dehosting_trimmed_R1.fastq.gz
<SAMPLE_ID>/<SAMPLE_ID>_pre-dehosting_trimmed_R2.fastq.gz

...to make clear that those reads have been trimmed, but not dehosted.

Dehosted Reads

If dehosting is performed, dehosted reads will be published under the directory supplied for the --outdir param, in a sub-directory for each sample, named using the sample ID. In addition, separate fastp reports will be generated before and after dehosting.

For example:

outdir
├── sample-01
│   ├── sample-01_dehosted_R1.fastq.gz
│   ├── sample-01_dehosted_R2.fastq.gz
│   ├── sample-01_post-dehosting_fastp.csv
│   ├── sample-01_post-dehosting_fastp.json
│   ├── sample-01_post-dehosting_fastp.html
│   ├── sample-01_pre-dehosting_fastp.csv
│   ├── sample-01_pre-dehosting_fastp.json
│   ├── sample-01_pre-dehosting_fastp.html
│   └── sample-01_hostile.log.json
├── sample-02
│   ├── sample-02_dehosted_R1.fastq.gz
│   ├── sample-02_dehosted_R2.fastq.gz
│   ├── sample-02_post-dehosting_fastp.csv
│   ├── sample-02_post-dehosting_fastp.json
│   ├── sample-02_post-dehosting_fastp.html
│   ├── sample-02_pre-dehosting_fastp.csv
│   ├── sample-02_pre-dehosting_fastp.json
│   ├── sample-02_pre-dehosting_fastp.html
│   └── sample-02_hostile.log.json
├── sample-03
│   ├── ...
...