CSHL_seqtec_2022 Live-coding Resource

Step 1: Get data

The data is hosted on DropBox, you can download it with the following wget command:
wget https://www.dropbox.com/s/pwjj61dzojg2ajy/T2T_data.tar.gz?dl=1 -O T2T_data.tar.gz

Unpack the data:
tar -xzf T2T_data.tar.gz

There are two directories in T2T_data:

1. alignments: contains chr21 alignments for two samples on GRCh38 and CHM13v2
2. references: contains fastas, gffs, and corresponding indicies for GRCh38 and CHM13v2

Where did the data come from?

1. CHM13v2.chr21.fasta: Full genome fasta downloaded from https://www.ncbi.nlm.nih.gov/assembly/GCA_009914755.4. Then, run samtools faidx <file> chr21 to isolate chr21.
2. GRCh38.chr21.fasta: Full genome fasta downloaded from https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.40. Then, run samtools faidx <file> chr21 to isolate chr21.
3. CHM13v2.chr21.gencode.v35.gff3.gz. Full GFF downloaded from https://github.com/marbl/CHM13#downloads (the Sorted GFF file under Assembly v2.0. Then, run tabix -p gff <file> and tabix <file> chr21 to isolate chr21.
4. GRCh38.chr21.gencode.v35.gff3.gz. Full GFF downloaded from https://www.gencodegenes.org/human/ under Comprehensive gene annotation. Then, run tabix -p gff <file> and tabix <file> chr21 to isolate chr21.
5. The CHM13v2 cram files were downloaded from the T2T AnVil workspace here. Navigate to the "Data" tab at the top, and then the "Participant" data table via the sidebar on the left. The crams and cram indicies are in the cram and cram_index columns, respectively. Then, run samtools view -ShC -T <ref.fasta> <cram> chr21 to isolate chr21. WARNING: The CRAM files are quite large (upwards of 10GB) and require an AnVilpayline to download.
6. The GRCh38 cram files were downloaded from SRA here. You'll have to get the SRA accession for each sample, and navigate to the appropriate directory in SRA. For example, sample HG00116 has accession: ERR3240132; the path to the cram file is https://ftp.sra.ebi.ac.uk/vol1/run/ERR324/ERR3240132/. Then, run samtools view -ShC -T <ref.fasta> <cram> chr21 to isolate chr21.

Step 2: Variant calling with freebayes

2.1: Run variant calling

Start a screen session to run CHM13v2 variant calling:
screen -S freebayes_CHM13v2

Within this screen session, run freebayes on the CHM13v2 alignments. We'll use time to keep track of how long variant calling took:
time freebayes -f T2T_data/references/CHM13v2.chr21.fasta --genotype-qualities T2T_data/alignments/*.CHM13v2.chr21.cram > CHM13v2.chr21.vcf

Start another screen session to run GRCh38 variant calling:
screen -S freebayes_GRCh38

Within this screen session, run freebayes on the GRCh38 alignments (using time as before):
time freebayes -f T2T_data/references/GRCh38.chr21.fasta --genotype-qualities T2T_data/alignments/*.GRCh38.chr21.cram > GRCh38.chr21.vcf

Once freebayes is done running, we shouldn't need these screen sessions anymore.

2.2: Filter to high-quality variants

Filter CHM13v2 vcf to variants with 99% probability:
vcffilter -f "QUAL > 20" CHM13v2.chr21.vcf > CHM13v2.chr21.filtered.vcf

Filter GRCh38 vcf to variants with 99% probability:
vcffilter -f "QUAL > 20" GRCh38.chr21.vcf > GRCh38.chr21.filtered.vcf

2.3: Zip and index the filtered vcfs

Zip the filtered CHM13v2 vcf:
bgzip CHM13v2.chr21.filtered.vcf
tabix -p vcf CHM13v2.chr21.filtered.vcf.gz

Zip the filtered GRCh38 vcf:
bgzip GRCh38.chr21.filtered.vcf
tabix -p vcf GRCh38.chr21.filtered.vcf.gz

Step 3: Get alignment statistics with samtools stats

Get alignment statistics for CHM13v2 bams:
samtools stats -r T2T_data/references/CHM13v2.chr21.fasta -@ 4 T2T_data/alignments/HG00116.CHM13v2.chr21.cram > HG00116.CHM13v2.chr21.cramstats.txt
samtools stats -r T2T_data/references/CHM13v2.chr21.fasta -@ 4 T2T_data/alignments/HG01509.CHM13v2.chr21.cram > HG01509.CHM13v2.chr21.cramstats.txt

Get alignment statistics for GRCh38 bams:
samtools stats -r T2T_data/references/GRCh38.chr21.fasta -@ 4 T2T_data/alignments/HG00116.GRCh38.chr21.cram > HG00116.GRCh38.chr21.cramstats.txt
samtools stats -r T2T_data/references/GRCh38.chr21.fasta -@ 4 T2T_data/alignments/HG01509.GRCh38.chr21.cram > HG01509.GRCh38.chr21.cramstats.txt

Step 4: Get variant calling statistics with bcftools stats

Get variant calling statistics for CHM13v2 vcf:
bcftools stats -s- CHM13v2.chr21.filtered.vcf.gz > CHM13v2.chr21.filtered.stats.txt

Get variant calling statistics for GRCh38 vcf:
bcftools stats -s- GRCh38.chr21.filtered.vcf.gz > GRCh38.chr21.filtered.stats.txt

Step 5: Plot alignment and variant calling statistics

Plot variant counts, as well as proper pairing, and mismatch rate stats for alignment, as shown in the analyze_stats.ipynb notebook.

Step 6: Plot KCNE1 alignments in IGV

First, either scp the crams to the local, or just wget them straight to the local.

Using grep determine the coordinates of KCNE1 from the Gencode gff files:
zgrep "KCNE1" CHM13v2.chr21.gencode.v35.gff3.gz | head -n 1 | cut -f 1,4,5
zgrep "KCNE1" GRCh38.chr21.gencode.v35.gff3.gz | head -n 1 | cut -f 1,4,5