INTRODUCTION

seqcap_pop is a series of commands and scripts we have been using for processing sequence capture data from population-level samples using tools within the Phyluce package (Faircloth 2015). This pipeline includes some additional scripts not available from Phyluce and its dependencies (in the "bin" folder). Also, it requires GATK v3 or above, which may not be included in the current Phyluce release.

LICENSE

The code within this repository is available under a 3-clause BSD license. See the License.txt file for more information.

CITATION

If you use this pipeline for your own research, please cite Phyluce following the instructions here and cite:

• Harvey MG, Smith BT, Glenn TC, Faircloth BC, Brumfield RT. 2016. Sequence capture versus restriction site associated DNA sequencing for shallow systematics. Systematic Biology 65: 910-924.

You can also provide a link to this repository if desired:

https://github.com/mgharvey/seqcap_pop

USAGE

Install Phyluce and dependencies following the instructions here. The custom Python scripts required for this pipeline are in the "bin" folder, so also download those and put them in your project folder. Make sure you have plenty of available hard drive space (depending on read counts, you may need upwards of 2GB for each sample). You will probably want to organize the output of each step below into a series of folders. Within my project folder, I typically set up a series of output folders labeled with consecutive numbers followed by a brief text descriptor:

• 1_raw-reads
• 2_clean-reads
• 3_velvet-output
• 4_match-contigs-to-probes
• 5_mapping
• 6_picard
• 7_merge-bams
• 8_GATK
• 9_SNP-tables
• 10_sequences
• 11_fasta-parts
• 12_raw-alignments
• 13_processed-phylip

You can make all of these folder now.

1. Clean raw reads (Illumiprocessor)

Follow the instructions in the Illumiprocessor documentation to make a configuration (.conf) file. Then execute Illumiprocessor. The command will look something like this:

illumiprocessor --input /path/to/1_raw-reads --output /path/to/2_clean-reads \
	--config illumiprocessor.conf --cores 8

2. Assemble reads into contigs (e.g., VelvetOptimiser)

In the past, I would combine reads from all of my individuals to make a consensus reference assembly (or, occasionally, use reads from the one individual with the highest quality data). Recently, I have been assembling the reads within each individual, then selecting the best contig for each target locus from among all the individual assemblies. I hope to find time to implement my own code for doing this, but I have been using code from Sonal Singhal's excellent Squamate Conserved Loci (SqCL) pipeline in the meantime. See the SqCL Github repository and paper for details.

Any of various assembly programs can be used for this step (see Phyluce documentation). I typically use VelvetOptimiser, which is not part of Phyluce, but can be obtained here. An example VelvetOptimiser command is:

VelvetOptimiser.pl --s 65 --e 75 --optFuncKmer=n50 --c=tbp -t 2 -a -f '-fastq.gz -shortPaired /path/to/2_clean-reads/Genus_species_1/split-adapter-quality-trimmed/Genus_species_1-READ1.fastq.gz /path/to/2_clean-reads/Genus_species_1/split-adapter-quality-trimmed/Genus_species_1-READ2.fastq.gz /path/to/2_clean-reads/Genus_species_2/split-adapter-quality-trimmed/Genus_species_2-READ1.fastq.gz /path/to/2_clean-reads/Genus_species_2/split-adapter-quality-trimmed/Genus_species_2-READ2.fastq.gz -short /path/to/2_clean-reads/Genus_species_1/split-adapter-quality-trimmed/Genus_species_1-READ-singleton.fastq.gz /path/to/2_clean-reads/Genus_species_2/split-adapter-quality-trimmed/Genus_species_2-READ-singleton.fastq.gz'

Other assemblers, however, should also work fine. Once you have a final contigs fasta file (this will be labeled "contigs.fa" if you used Velvet or VelvetOptimiser), put that in the "3_velvet-output" folder.

3. Map contigs to probes (Phyluce)

You next need to see which of the contigs that were assembled correspond to the loci targeted in your sequence capture procedure. You do this by mapping the contigs to the sequences from the probes you used. When your assemblies are from multiple individuals, particularly in species containing high levels of genetic structure, you are likely to obtain multiple contigs from the same locus (because some individuals are very divergent from others at that genomic position). Typically, Phyluce would dispose of contigs in situations where multiple contigs map to the same locus, so you would lose a lot of loci at this step. I have tweaked a script from Brant Faircloth and Graham Derryberry (extract_uce_bypass_MGH.py) so as to save one contig in situations where multiple contigs map to a single locus. If you have used multiple individuals to make your assembly, you may want to use this script. If you have used only one, you will probably be fine using the original script (extract_uce_bypass.py). The commands will look like this:

If multiple individuals in assembly:

python extract_uce_bypass_MGH.py \
	/path/to/3_velvet-output/Genus_species/contigs.fa \
	/path/to/probe_sequences.fasta \
	/path/to/4_match-contigs-to-probes/Genus_species.fasta \
	/path/to/4_match-contigs-to-probes/Genus_species.lastz

If one individual in assembly:

python extract_uce_bypass.py \
	/path/to/3_velvet-output/Genus_species/contigs.fa \
	/path/to/probe_sequences.fasta \
	/path/to/4_match-contigs-to-probes/Genus_species.fasta \
	/path/to/4_match-contigs-to-probes/Genus_species.lastz

4. Map reads to contigs (BWA)

Next, we map the reads back to the contigs to obtain a pileup. This is the most finicky step of the pipeline, as BWA often fails. I have 3 different versions of BWA installed, and if a sample repeatedly fails in one version, I switch to a different version (algorithms differ slightly across versions). I typically have success with BWA 0.7.4, 0.7.3, or 0.7.0.

For individual 1:

bwa index -a is /path/to/4_match-contigs-to-probes/Genus_species.fasta

bwa aln /path/to/4_match-contigs-to-probes/Genus_species.fasta \
	/path/to/2_clean-reads/Genus_species_1/split-adapter-quality-trimmed/Genus_species_1-READ1.fastq.gz \ 
	> /path/to/5-mapping/Genus_species_1_read1.sa.sai

bwa aln /path/to/4_match-contigs-to-probes/Genus_species.fasta \
	/path/to/2_clean-reads/Genus_species_1/split-adapter-quality-trimmed/Genus_species_1-READ2.fastq.gz  \
	> /path/to/5-mapping/Genus_species_1_read2.sa.sai

bwa sampe /path/to/4_match-contigs-to-probes/Genus_species.fasta \
	/path/to/5-mapping/Genus_species_1_read1.sa.sai /path/to/5-mapping/Genus_species_1_read2.sa.sai \
	/path/to/2_clean-reads/Genus_species_1/split-adapter-quality-trimmed/Genus_species_1-READ1.fastq.gz \
	/path/to/2_clean-reads/Genus_species_1/split-adapter-quality-trimmed/Genus_species_1-READ2.fastq.gz \
	> /path/to/5-mapping/Genus_species_1-aln.sam

Then do the same thing for individual 2 (and any additional individuals).

5. Convert .sam file to .bam file (samtools)

samtools view -bS /path/to/5-mapping/Genus_species_1-aln.sam \
	> /path/to/5-mapping/Genus_species_1-aln.bam

Then do the same thing for individual 2 (and any additional individuals).

6. Clean the .bam file (Picard)

This step soft-clips reads at the end of the reference contigs.

java -jar ~/anaconda/jar/CleanSam.jar \
	I=/path/to/5-mapping/Genus_species_1-aln.bam \
	O=/path/to/6_picard/Genus_species_1-aln_CL.bam \
	VALIDATION_STRINGENCY=SILENT

Then do the same thing for individual 2 (and any additional individuals).

7. Add read groups (Picard)

java -Xmx2g -jar ~/anaconda/jar/AddOrReplaceReadGroups.jar \
    I=/path/to/6_picard/Genus_species_1-aln_CL.bam \
    O=/path/to/6_picard/Genus_species_1-aln_RG.bam \
    SORT_ORDER=coordinate \
    RGPL=illumina \
    RGPU=TestXX \
    RGLB=Lib1 \
    RGID=Genus_species_1 \
    RGSM=Genus_species_1 \
    VALIDATION_STRINGENCY=LENIENT

Then do the same thing for individual 2 (and any additional individuals).

8. Mark PCR duplicate reads (Picard)

java -Xmx2g -jar ~/anaconda/jar/MarkDuplicates.jar \
    I=/path/to/6_picard/Genus_species_1-aln_RG.bam \
    O=/path/to/6_picard/Genus_species_1-aln_MD.bam \
    METRICS_FILE=/path/to/6_picard/Genus_species_1.metrics \
    MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=250 \
    ASSUME_SORTED=true \
    REMOVE_DUPLICATES=false

Then do the same thing for individual 2 (and any additional individuals).

9. Merge the BAM files across individuals within your species (Phyluce)

java -Xmx2g -jar ~/anaconda/jar/MergeSamFiles.jar \
    SO=coordinate \
    AS=true \
    I=/path/to/6_picard/Genus_species_1-aln_MD.bam \
    I=/path/to/6_picard/Genus_species_2-aln_MD.bam \
    O=/path/to/7_merge-bams/Genus_species.bam 

10. Index the merged .bam file (samtools)

samtools index /Volumes/G-DRIVE/Amazon/7_merge-bams/Genus_species.bam 

11. Create a dictionary from the reference contigs (Picard)

java -Xmx2g -jar ~/anaconda/pkgs/picard-1.106-0/jar/CreateSequenceDictionary.jar \
    R=/path/to/4_match-contigs-to-probes/Genus_species.fasta \
    O=/path/to/4_match-contigs-to-probes/Genus_species.dict

12. Index the reference (samtools)

samtools faidx /path/to/4_match-contigs-to-probes/Genus_species.fasta

13. Call indels (GATK)

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T RealignerTargetCreator \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -I /path/to/7_merge-bams/Genus_species.bam  \
    --minReadsAtLocus 7 \
    -o /path/to/8_GATK/Genus_species.intervals

14. Realign indels (GATK)

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T IndelRealigner \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -I /path/to/7-merge/Genus_species.bam  \
    -targetIntervals /path/to/8_GATK/Genus_species.intervals \
    -LOD 3.0 \
    -o /path/to/8_GATK/Genus_species_RI.bam

15. Call SNPs (GATK)

Here I have ploidy set to 2. This can be problematic when some data comes from the sex chromosome(s) and we have heterogametic individuals. It might make sense to map loci to an existing genome, and either filter out sex-linked loci, or bring them through this part of the pipeline separately from autosomal loci. Another problem, at least in birds, is that the W chromosome (present only in females) is not always very degenerate, and retains some of the conserved loci we are targeting on the Z chromosome. This results in sequence contamination from the W chromosome in some of the sequence alignments with female individuals. These can be filtered out after the fact (by looking for female heterozygotes at Z-linked loci), but there may be better solutions. More work needed here.

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T UnifiedGenotyper \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -I /path/to/8_GATK/Genus_species_RI.bam \
    -gt_mode DISCOVERY \
    -o /path/to/8_GATK/Genus_species_raw_SNPs.vcf \
    -ploidy 2 \
    -rf BadCigar

16. Annotate SNPs (GATK)

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T VariantAnnotator \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -I /path/to/8_GATK/Genus_species_RI.bam \
    -G StandardAnnotation \
    -V:variant,VCF /path/to/8_GATK/Genus_species_raw_SNPs.vcf \
    -XA SnpEff \
    -o /path/to/8_GATK/Genus_species_SNPs_annotated.vcf \
    -rf BadCigar      

17. Annotate Indels (GATK)

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T UnifiedGenotyper \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -I /path/to/8_GATK/Genus_species_RI.bam \
    -gt_mode DISCOVERY \
    -glm INDEL \
    -o /path/to/8_GATK/Genus_species_SNPs_indels.vcf \
    -rf BadCigar         

18. Mask indels (GATK)

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T VariantFiltration \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -V /path/to/8_GATK/Genus_species_raw_SNPs.vcf \
    --mask /path/to/8_GATK/Genus_species_SNPs_indels.vcf \
    --maskExtension 5 \
    --maskName InDel \
    --clusterWindowSize 10 \
    --filterExpression "MQ0 >= 4 && ((MQ0 / (1.0 * DP)) > 0.1)" \
    --filterName "BadValidation" \
    --filterExpression "QUAL < 30.0" \
    --filterName "LowQual" \
    --filterExpression "QD < 5.0" \
    --filterName "LowVQCBD" \
    -o /path/to/8_GATK/Genus_species_SNPs_no_indels.vcf  \
    -rf BadCigar

19. Restrict to high-quality SNPs (bash)

cat /path/to/8_GATK/Genus_species_SNPs_no_indels.vcf | grep 'PASS\|^#' > /path/to/8_GATK/Genus_species_SNPs_pass-only.vcf 

20. Read-backed phasing (GATK)

The output of this step is a .vcf file containing the final set of phased SNPs for all individuals. This can then be used to produce input files for SNP-based analyses. The subsequent two steps (21 and 22) also produce output containing these same SNPs, but separated into a file for each individual.

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T ReadBackedPhasing \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -I /path/to/8_GATK/Genus_species_RI.bam \
    --variant /path/to/8_GATK/Genus_species_SNPs_pass-only.vcf \
    -L /path/to/8_GATK/Genus_species_SNPs_pass-only.vcf \
    -o /path/to/8_GATK/Genus_species_SNPs_phased.vcf \
    --phaseQualityThresh 20.0 \
    -rf BadCigar

21. Make a vcf for each sample (GATK)

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -T SelectVariants \
    --variant /path/to/8_GATK/Genus_species_SNPs_phased.vcf \
    -o /path/to/8_GATK/Genus_species_1_SNPs.vcf \
    -sn Genus_species_1 \
    -rf BadCigar

Then do the same thing for individual 2 (and any additional individuals).

22. Make a table of phased SNPs for each sample (GATK)

java -Xmx2g -jar ~/anaconda/GenomeAnalysisTK-3.3-0/GenomeAnalysisTK.jar \
    -T VariantsToTable \
    -R /path/to/4_match-contigs-to-probes/Genus_species.fasta \
    -V /path/to/8_GATK/Genus_species_1_SNPs.vcf \
    -F CHROM -F POS -F QUAL -GF GT -GF DP -GF HP -GF AD \
    -o /path/to/9_SNP-tables/Genus_species_1_SNPs_phased-table.txt \
    -rf BadCigar

Then do the same thing for individual 2 (and any additional individuals).

23. Add phased SNPs to reference and optionally filter (seqcap_pop script)

Because GATK does not output sequences or SNP calls for invariant sites, we need to add the SNPs back into the reference sequences if we want alignments. This is not optimal, as sequence data will be present at invariant sites even if no reads inform that site for a given individual. However, this is currently the only way I can find to take advantage of read-backed phasing and get the phased calls into alignment format. Freebayes allows output of invariant and variant sites, which can then easily be used to produce complete alignments. However, the phasing options in Freebayes are much more limited, and there is no obvious way to use Freebayes to output data once SNPs have been phased in GATK. If you want to go the Freebayes route, a script to obtain alignments from .vcf Freebayes output is here. This part of the pipeline could obviously use some improvement!

python add_phased_snps_to_seqs_filter.py \
	/path/to/4_match-contigs-to-probes/Genus_species.fasta \
	/path/to/9_SNP-tables/Genus_species_1_SNPs_phased-table.txt \
	/path/to/10_sequences/Genus_species/Genus_species_1_sequences.txt \
	1

Then do the same thing for individual 2 (and any additional individuals).

The final argument ("1") above filters out any alleles not supported by a particular number of reads. This can be increased in order to set a hard filter on the minimum number of reads for allele calls (generally not recommended).

24. Collate sequences from all individuals into files by UCE (seqcap_pop script)

python collate_sample_fastas_GATK.py \
	/path/to/10_sequences/Genus_species/ \
	/path/to/11_fasta-parts/Genus_species/ \
	sequences.txt

25. Align the sequences (MAFFT)

Although the sequences should line up, this serves to guarantee that there won't be any issues.

python run_mafft.py \
	/path/to/11_fasta-parts/Genus_species/ \
	/path/to/12_raw-alignments/Genus_species/

26. Process the alignments (seqcap_pop script)

This makes phylip alignments from the raw fasta files from MAFFT.

python process_mafft_alignments_GATK.py \
	/path/to/12_raw-alignments/Genus_species \
	/path/to/13_processed-phylip/Genus_species

File format conversion

For programs that don't accept .vcf files of SNPs or phylip sequence alignments, there are some additional scripts in the "bin" folder that permit data format conversion (I will be adding more here as I write them):

• structure_from_vcf.py should produce files of all SNPs containing linkage and phasing information for use in STRUCTURE.

python structure_from_vcf.py \
	/path/to/8_GATK/Genus_species_SNPs_phased.vcf \
	/path/to/desired/output/directory/Genus_species_STRUCTURE.txt

• faststructure_from_vcf.py should produce files of all SNPs, the first SNP per locus, or a random SNP from each locus for use in fastSTRUCTURE.

python faststructure_from_vcf.py \
	/path/to/8_GATK/Genus_species_SNPs_phased.vcf \
	/path/to/desired/output/directory/ \
	Genus_species \
	--all \
	--first \
	--random

• adegenet_from_vcf.py should produce files of all SNPs, the first SNP per locus, or a random SNP from each locus for use in the R package adegenet (which can do PCAs, discriminant analysis of principle componenents, etc.).

python adegenet_from_vcf.py \
	/path/to/8_GATK/Genus_species_SNPs_phased.vcf \
	/path/to/desired/output/directory/ \
	Genus_species \
	--all \
	--first \
	--random

• genepop_from_vcf.py should produce files of all SNPs, the first SNP per locus, or a random SNP from each locus for use in GenePop.

python genepop_from_vcf.py \
	/path/to/8_GATK/Genus_species_SNPs_phased.vcf \
	/path/to/desired/output/directory/ \
	Genus_species \
	--all \
	--first \
	--random

• gphocs_from_phy.py reformats alignments for input into G-PhoCS.

python gphocs_from_phy.py \
	/path/to/13_processed-phylip \
	/path/to/desired/output/directory/Genus_species_GPHOCS.txt