examples/human_variation_aware_alignment/Analysis.ipynb

{
 "cells": [
  {
   "cell_type": "markdown",
   "id": "b21c6a85-d536-45a7-8531-3a7d5472ffb1",
   "metadata": {},
   "source": [
    "The human reference genome is intended to be a linear consensus sequence of many individuals. This can influence mapping accuracy, as natural variation of an individual (due to ethniticy, etc.) will reduce alignment scores and thus suppress reads in certain areas. This can influence variant calling, as reads containing both known and novel variation can score low enough to be omitted by mapping software.\n",
    "\n",
    "To address this, we encode the known variation sites into the reference genome and use a graph aligner. We will use genome in a bottle's dataset and a graph aligner for analysis.\n",
    "\n",
    "This was ran on a machine with 128GB of memory and 32 cores (m5.8xlarge) and 2 tb disk."
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 49,
   "id": "c6e1f3b7-2280-497e-8bca-6c289843915f",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "--2024-10-03 11:32:04--  https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/release/NA12878_HG001/NISTv4.2.1/GRCh38/HG001_GRCh38_1_22_v4.2.1_benchmark.vcf.gz\n",
      "Resolving ftp-trace.ncbi.nlm.nih.gov (ftp-trace.ncbi.nlm.nih.gov)... 130.14.250.13, 130.14.250.7, 2607:f220:41e:250::7, ...\n",
      "Connecting to ftp-trace.ncbi.nlm.nih.gov (ftp-trace.ncbi.nlm.nih.gov)|130.14.250.13|:443... connected.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 125932193 (120M) [application/x-gzip]\n",
      "Saving to: ‘HG001_GRCh38_1_22_v4.2.1_benchmark.vcf.gz’\n",
      "\n",
      "HG001_GRCh38_1_22_v 100%[===================>] 120.10M  6.49MB/s    in 16s     \n",
      "\n",
      "2024-10-03 11:32:20 (7.46 MB/s) - ‘HG001_GRCh38_1_22_v4.2.1_benchmark.vcf.gz’ saved [125932193/125932193]\n",
      "\n",
      "--2024-10-03 11:32:20--  https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz\n",
      "Resolving hgdownload.soe.ucsc.edu (hgdownload.soe.ucsc.edu)... 128.114.119.163\n",
      "Connecting to hgdownload.soe.ucsc.edu (hgdownload.soe.ucsc.edu)|128.114.119.163|:443... connected.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 983659424 (938M) [application/x-gzip]\n",
      "Saving to: ‘hg38.fa.gz.1’\n",
      "\n",
      "hg38.fa.gz.1         28%[====>               ] 267.98M  3.74MB/s    eta 2m 15s ^C\n"
     ]
    }
   ],
   "source": [
    "# for analysis, we'll be using genome in a bottle, so we use their known variants. This could just as easily be dbsnp/etc.\n",
    "!wget https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/release/NA12878_HG001/NISTv4.2.1/GRCh38/HG001_GRCh38_1_22_v4.2.1_benchmark.vcf.gz\n",
    "!wget https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 41,
   "id": "63ad3726-9c98-4388-81ad-da4200a2730a",
   "metadata": {},
   "outputs": [],
   "source": [
    "# we just do chromosome 1 to make things a bit faster for this example\n",
    "!samtools faidx hg38.fa.gz chr1 | bgzip -c - > chr1.fa.gz\n",
    "!samtools faidx chr1.fa.gz"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 50,
   "id": "00958bd4-9197-483d-a38a-1c9aab875726",
   "metadata": {},
   "outputs": [],
   "source": [
    "!gunzip -c HG001_GRCh38_1_22_v4.2.1_benchmark.vcf.gz | awk '/^#/ || /^chr1\\t/' | bgzip -c > chr1.vcf.bgz"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 85,
   "id": "4a5c879b-5d70-435e-afae-ccfc16299338",
   "metadata": {},
   "outputs": [],
   "source": [
    "# cleanup any old stuff\n",
    "!rm -rf .gen\n",
    "!rm hg38.db"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 86,
   "id": "c791680b-d5ed-438a-a514-f675dcb2acc7",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "Gen repository initialized.\n",
      "Created it\n"
     ]
    }
   ],
   "source": [
    "!gen init\n",
    "!gen --db hg38.db import --name hg38 --fasta ./chr1.fa.gz --shallow"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 87,
   "id": "01764e02-2efc-4d9e-bd95-ceec37a14280",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
     ]
    }
   ],
   "source": [
    "!gen --db hg38.db update --name hg38 --vcf chr1.vcf.bgz"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 5,
   "id": "ef0d0618-c322-4490-b6cd-dd34d4c19e05",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "--2024-10-02 10:47:00--  https://github.com/vgteam/vg/releases/download/v1.60.0/vg\n",
      "Resolving github.com (github.com)... 140.82.112.4\n",
      "Connecting to github.com (github.com)|140.82.112.4|:443... connected.\n",
      "HTTP request sent, awaiting response... 302 Found\n",
      "Location: https://objects.githubusercontent.com/github-production-release-asset-2e65be/24727800/76d354fd-0852-4676-b3af-82df87da06a0?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=releaseassetproduction%2F20241002%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20241002T144701Z&X-Amz-Expires=300&X-Amz-Signature=e89751dbe24c5a1e1c176bd81248d12522051a3498f0100e80fd5b72d6b87554&X-Amz-SignedHeaders=host&response-content-disposition=attachment%3B%20filename%3Dvg&response-content-type=application%2Foctet-stream [following]\n",
      "--2024-10-02 10:47:00--  https://objects.githubusercontent.com/github-production-release-asset-2e65be/24727800/76d354fd-0852-4676-b3af-82df87da06a0?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=releaseassetproduction%2F20241002%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20241002T144701Z&X-Amz-Expires=300&X-Amz-Signature=e89751dbe24c5a1e1c176bd81248d12522051a3498f0100e80fd5b72d6b87554&X-Amz-SignedHeaders=host&response-content-disposition=attachment%3B%20filename%3Dvg&response-content-type=application%2Foctet-stream\n",
      "Resolving objects.githubusercontent.com (objects.githubusercontent.com)... 185.199.110.133, 185.199.109.133, 185.199.108.133, ...\n",
      "Connecting to objects.githubusercontent.com (objects.githubusercontent.com)|185.199.110.133|:443... connected.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 46259960 (44M) [application/octet-stream]\n",
      "Saving to: ‘vg’\n",
      "\n",
      "vg                  100%[===================>]  44.12M  53.4MB/s    in 0.8s    \n",
      "\n",
      "2024-10-02 10:47:01 (53.4 MB/s) - ‘vg’ saved [46259960/46259960]\n",
      "\n"
     ]
    }
   ],
   "source": [
    "# now, we want to align using vg\n",
    "!wget https://github.com/vgteam/vg/releases/download/v1.60.0/vg\n",
    "!chmod +x ./vg"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 8,
   "id": "6ac133f7-5f47-4f4a-90de-f988382ba2ef",
   "metadata": {},
   "outputs": [],
   "source": [
    "!gen --db hg38.db export --name hg38 --gfa hg38.gfa"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 20,
   "id": "bc61d6f7-9f4c-4b2f-8cb5-1875573cb4b9",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "--2024-10-02 16:35:58--  https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_001.fastq.gz\n",
      "Resolving ftp-trace.ncbi.nlm.nih.gov (ftp-trace.ncbi.nlm.nih.gov)... 130.14.250.13, 130.14.250.7, 2607:f220:41e:250::13, ...\n",
      "Connecting to ftp-trace.ncbi.nlm.nih.gov (ftp-trace.ncbi.nlm.nih.gov)|130.14.250.13|:443... connected.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 432595315 (413M) [application/x-gzip]\n",
      "Saving to: ‘U5b_AGTTCC_L001_R1_001.fastq.gz’\n",
      "\n",
      "U5b_AGTTCC_L001_R1_ 100%[===================>] 412.55M  11.5MB/s    in 35s     \n",
      "\n",
      "2024-10-02 16:36:34 (11.7 MB/s) - ‘U5b_AGTTCC_L001_R1_001.fastq.gz’ saved [432595315/432595315]\n",
      "\n",
      "--2024-10-02 16:36:34--  https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_001.fastq.gz\n",
      "Resolving ftp-trace.ncbi.nlm.nih.gov (ftp-trace.ncbi.nlm.nih.gov)... 130.14.250.13, 130.14.250.7, 2607:f220:41e:250::13, ...\n",
      "Connecting to ftp-trace.ncbi.nlm.nih.gov (ftp-trace.ncbi.nlm.nih.gov)|130.14.250.13|:443... connected.\n",
      "HTTP request sent, awaiting response... 200 OK\n",
      "Length: 469449584 (448M) [application/x-gzip]\n",
      "Saving to: ‘U5b_AGTTCC_L001_R2_001.fastq.gz’\n",
      "\n",
      "U5b_AGTTCC_L001_R2_ 100%[===================>] 447.70M  12.7MB/s    in 37s     \n",
      "\n",
      "2024-10-02 16:37:11 (12.1 MB/s) - ‘U5b_AGTTCC_L001_R2_001.fastq.gz’ saved [469449584/469449584]\n",
      "\n"
     ]
    }
   ],
   "source": [
    "# for alignment, a random genome in a bottle sample\n",
    "!wget -O R1.fq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_001.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_002.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_003.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_004.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_005.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_006.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_007.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R1_008.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R1_001.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R1_002.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R1_003.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R1_004.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R1_005.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R1_006.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R1_007.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R1_008.fastq.gz\n",
    "!wget -O R2.fq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_001.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_002.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_003.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_004.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_005.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_006.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_007.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L001_R2_008.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R2_001.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R2_002.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R2_003.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R2_004.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R2_005.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R2_006.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R2_007.fastq.gz https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/NIST_NA12878_HG001_HiSeq_300x/140313_D00360_0014_AH8GGVADXX/Project_RM8398/Sample_U5b/U5b_AGTTCC_L002_R2_008.fastq.gz"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "id": "4c8fc337-dcc2-4798-a6df-3355e7645844",
   "metadata": {},
   "outputs": [],
   "source": [
    "# This builds our indices up\n",
    "!./vg mod -X 32 hg38.gfa > hg38.mod.gfa\n",
    "!./vg autoindex -p index -w map -g hg38.mod.gfa"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "id": "f77c8ae5-a389-468c-8a61-471bfa4e091e",
   "metadata": {},
   "outputs": [],
   "source": [
    "# this maps reads\n",
    "!./vg map -x index.xg -g index.gcsa -f R1.fq.gz -f R2.fq.gz > align.gam\n",
    "!./vg gamsort -p align.gam > align.sorted.gam\n",
    "!./vg index -l align.sorted.gam"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "id": "e24fcaf3-d878-4335-97b7-cdd43993981d",
   "metadata": {},
   "outputs": [],
   "source": [
    "# this summarizes our reads per position and then calls variants\n",
    "!./vg pack -e -x index.xg -g align.sorted.gam -o aln.pack\n",
    "!./vg call index.xg -k aln.pack > align.vcf"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 2,
   "id": "3a5e2355-8e4a-4ac9-b067-710bf8f59a23",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "chr1\t783006\t.\tA\tG\t50\tPASS\tplatforms=4;platformnames=PacBio,Illumina,10X,CG;datasets=4;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR,CGnormal;callsets=6;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,CGnormal,HiSeqPE300xfreebayes;datasetsmissingcall=IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_CGnormal_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:652:16,234:0,82:312\n",
      "chr1\t783175\t.\tT\tC\t50\tPASS\tplatforms=4;platformnames=PacBio,Illumina,10X,Solid;datasets=4;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR,SolidSE75bp;callsets=6;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes,SolidSE75GATKHC;datasetsmissingcall=CGnormal,IonExome;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:639:0,218:0,84:194\n",
      "chr1\t784860\t.\tT\tC\t50\tPASS\tplatforms=4;platformnames=PacBio,Illumina,10X,CG;datasets=4;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR,CGnormal;callsets=6;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,CGnormal,HiSeqPE300xfreebayes;datasetsmissingcall=IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_CGnormal_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:901:105,406:0,74:301\n",
      "chr1\t785417\t.\tG\tA\t50\tPASS\tplatforms=4;platformnames=PacBio,Illumina,10X,CG;datasets=4;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR,CGnormal;callsets=6;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,CGnormal,HiSeqPE300xfreebayes;datasetsmissingcall=IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_CGnormal_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:820:125,383:0,70:339\n",
      "chr1\t797392\t.\tG\tA\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_10XLRGATK_filt,CS_CCS15kb_20kbGATK4_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t0/1:.:760:161,142:25,37:147\n",
      "chr1\t798618\t.\tC\tT\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_10XLRGATK_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:526:0,196:0,65:157\n",
      "chr1\t798662\t.\tG\tA\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_10XLRGATK_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:563:0,211:0,65:147\n",
      "chr1\t800046\t.\tG\tA\t50\tPASS\tplatforms=4;platformnames=PacBio,Illumina,10X,CG;datasets=4;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR,CGnormal;callsets=6;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,CGnormal,HiSeqPE300xfreebayes;datasetsmissingcall=IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_CGnormal_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:640:21,249:0,68:197\n",
      "chr1\t801142\t.\tA\tT\t50\tPASS\tplatforms=4;platformnames=PacBio,Illumina,10X,CG;datasets=4;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR,CGnormal;callsets=6;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,CGnormal,HiSeqPE300xfreebayes;datasetsmissingcall=IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_CGnormal_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:431:51,183:0,67:246\n",
      "chr1\t801143\t.\tT\tC\t50\tPASS\tplatforms=4;platformnames=PacBio,Illumina,10X,CG;datasets=4;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR,CGnormal;callsets=6;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,CGnormal,HiSeqPE300xfreebayes;datasetsmissingcall=IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_CGnormal_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:431:51,183:0,67:249\n",
      "chr1\t804046\t.\tA\tG\t50\tPASS\tplatforms=4;platformnames=PacBio,Illumina,10X,CG;datasets=4;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR,CGnormal;callsets=6;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,CGnormal,HiSeqPE300xfreebayes;datasetsmissingcall=IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:639:47,254:0,78:334\n",
      "chr1\t804148\t.\tG\tA\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:612:0,208:0,79:156\n",
      "chr1\t805514\t.\tAC\tA\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_10XLRGATK_filt,CS_CCS15kb_20kbGATK4_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t0/1:.:712:142,135:40,42:151\n",
      "chr1\t806199\t.\tT\tC\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbGATK4,HiSeqPE300xGATK,CCS15kb_20kbDV,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_10XLRGATK_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:529:0,161:0,76:197\n",
      "chr1\t807445\t.\tA\tG\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_10XLRGATK_filt,CS_CCS15kb_20kbGATK4_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t0/1:.:773:136,148:38,51:150\n",
      "chr1\t807641\t.\tT\tC\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_10XLRGATK_filt,CS_CCS15kb_20kbGATK4_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t0/1:.:753:157,111:51,37:158\n",
      "chr1\t807692\t.\tC\tA\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_10XLRGATK_filt,CS_CCS15kb_20kbGATK4_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:802:15,283:0,87:162\n",
      "chr1\t813498\t.\tG\tT\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:798:3,298:0,90:161\n",
      "chr1\t813885\t.\tG\tA\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:615:0,204:0,85:161\n",
      "chr1\t814016\t.\tT\tG\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_10XLRGATK_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t0/1:.:582:100,94:38,47:157\n",
      "chr1\t814033\t.\tC\tT\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_10XLRGATK_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t0/1:.:574:97,91:39,47:158\n",
      "chr1\t814212\t.\tG\tA\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:289:0,45:0,88:162\n",
      "chr1\t814264\t.\tT\tC\t50\tPASS\tplatforms=2;platformnames=PacBio,10X;datasets=2;datasetnames=CCS15kb_20kb,10XChromiumLR;callsets=3;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,10XLRGATK;datasetsmissingcall=HiSeqPE300x,CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:197:0,89:0,89:68\n",
      "chr1\t814303\t.\tC\tG\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:330:0,61:0,88:158\n",
      "chr1\t814309\t.\tT\tG\t50\tPASS\tplatforms=3;platformnames=PacBio,Illumina,10X;datasets=3;datasetnames=CCS15kb_20kb,HiSeqPE300x,10XChromiumLR;callsets=5;callsetnames=CCS15kb_20kbDV,CCS15kb_20kbGATK4,HiSeqPE300xGATK,10XLRGATK,HiSeqPE300xfreebayes;datasetsmissingcall=CGnormal,IonExome,SolidSE75bp;callable=CS_CCS15kb_20kbDV_callable,CS_CCS15kb_20kbGATK4_callable;filt=CS_HiSeqPE300xGATK_filt,CS_HiSeqPE300xfreebayes_filt;difficultregion=HG001.hg38.300x.bam.bilkentuniv.010920.dups,hg38.segdups_sorted_merged,lowmappabilityall\tGT:PS:DP:ADALL:AD:GQ\t1/1:.:338:0,65:1,89:158\n",
      "grep: write error: Broken pipe\n",
      "\n",
      "gzip: stdout: Broken pipe\n"
     ]
    }
   ],
   "source": [
    "# look at some variants from our data vs. input\n",
    "!gunzip -c chr1.vcf.bgz | grep -P \"chr1\\t\" | head -n 25"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 6,
   "id": "a0fa600a-2425-4792-8841-ec00ffee01b9",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "1-chr1\t783006\t>126167>126169\tA\tG\t220.146\tPASS\tAT=>126167>126168>126169,>126167>8632817>126169;DP=38\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:38:24,14:-26.597657,-5.060118,-49.472423:215:-1.098612:21.203342:14\n",
      "1-chr1\t783175\t>126174>126176\tT\tC\t24.9355\tPASS\tAT=>126174>126175>126176,>126174>8532755>126176;DP=27\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:27:23,4:-8.152426,-6.140191,-51.312071:20:-1.108288:17.000000:4\n",
      "1-chr1\t784860\t>126228>126230\tT\tC\t137.863\tPASS\tAT=>126228>126229>126230,>126228>8500678>126230;DP=6\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:6:0,6:-14.601243,-2.614278,-1.313245:13:-1.147402:5.849057:6\n",
      "1-chr1\t785417\t>126247>126249\tG\tA\t183.11\tPASS\tAT=>126247>126248>126249,>126247>74686>126249;DP=8\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:8:0,8:-19.265059,-3.282439,-1.437336:18:-1.112797:8.246754:8\n",
      "1-chr1\t797392\t>126623>126625\tG\tA\t16.7555\tPASS\tAT=>126623>126624>126625,>126623>55966>126625;DP=4\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:4:3,1:-3.256084,-2.086064,-7.427831:11:-1.164035:9.014085:1\n",
      "1-chr1\t798618\t>126663>126665\tC\tT\t251.478\tPASS\tAT=>126663>126664>126665,>126663>8352225>126665;DP=19\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:19:6,13:-27.052184,-2.381459,-10.978723:85:-1.098612:20.113207:6\n",
      "1-chr1\t798662\t>126666>126668\tG\tA\t274.171\tPASS\tAT=>126666>126667>126668,>126666>66225>126668;DP=12\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:12:0,12:-29.002329,-5.028399,-2.062721:30:-1.099550:17.687372:12\n",
      "1-chr1\t800046\t>126711>126713\tG\tA\t288.775\tPASS\tAT=>126711>126712>126713,>126711>19634>126713;DP=20\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:20:5,15:-31.376685,-2.976278,-9.015825:59:-1.791761:22.250000:5\n",
      "1-chr1\t804046\t>126840>126842\tA\tG\t204.417\tPASS\tAT=>126840>126841>126842,>126840>8609705>126842;DP=9\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:9:0,9:-21.641444,-3.660997,-1.681302:19:-1.109037:7.611966:9\n",
      "1-chr1\t804148\t>126845>126847\tG\tA\t171.565\tPASS\tAT=>126845>126846>126847,>126845>37961>126847;DP=15\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:15:6,9:-18.556125,-1.876711,-11.637447:97:-1.098612:13.428572:6\n",
      "1-chr1\t805514\t>126889>126891\tAC\tA\t131.609\tPASS\tAT=>126889>126890>126891,>126889>71794>126891;DP=13\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:13:6,7:-14.391369,-1.707609,-11.890158:101:-1.098612:14.642366:6\n",
      "1-chr1\t806199\t>126912>126914\tT\tC\t251.278\tPASS\tAT=>126912>126913>126914,>126912>8516579>126914;DP=11\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:11:0,11:-26.266138,-4.290036,-1.616284:6:-1.289976:12.315789:11\n",
      "1-chr1\t807445\t>126952>126954\tA\tG\t26.3073\tPASS\tAT=>126952>126953>126954,>126952>8621004>126954;DP=10\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:10:8,2:-4.441830,-2.291278,-17.972867:21:-1.105658:10.683616:2\n",
      "1-chr1\t807641\t>126960>126962\tT\tC\t93.1057\tPASS\tAT=>126960>126961>126962,>126960>8528873>126962;DP=10\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:10:5,5:-10.411785,-1.578314,-10.411785:88:-1.098612:8.629630:5\n",
      "1-chr1\t807692\t>126963>126965\tC\tA\t57.2268\tPASS\tAT=>126963>126964>126965,>126963>88148>126965;DP=6\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:6:3,3:-6.627907,-1.382339,-6.627907:58:-1.098615:7.499431:3\n",
      "1-chr1\t813498\t>127146>127148\tG\tT\t111.63\tPASS\tAT=>127146>127147>127148,>127146>8385505>127148;DP=12\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:12:6,6:-12.367268,-1.681336,-12.367268:106:-1.098612:10.179487:6\n",
      "1-chr1\t813885\t>127160>127162\tG\tA\t18.1932\tPASS\tAT=>127160>127161>127162,>127160>13182>127162;DP=13\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:13:11,2:-4.338595,-3.016609,-24.691681:13:-1.145157:12.313433:2\n",
      "1-chr1\t814016\t>127166>127168\tT\tG\t224.653\tPASS\tAT=>127166>127167>127168,>127166>8657170>127168;DP=28\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:28:15,13:-24.161733,-2.173510,-28.673428:219:-1.098612:24.058823:13\n",
      "1-chr1\t814033\t>127168>127170\tC\tT\t154.919\tPASS\tAT=>127168>127169>127170,>127168>8405985>127170;DP=21\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:21:12,9:-17.017011,-2.002242,-23.515110:150:-1.098612:23.060181:9\n",
      "1-chr1\t814212\t>127175>127177\tG\tA\t118.037\tPASS\tAT=>127175>127176>127177,>127175>10274>127177;DP=5\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:5:0,5:-13.532101,-3.542964,-2.225898:86:-1.098612:12.513846:5\n",
      "1-chr1\t814264\t>127178>127180\tT\tC\t86.2114\tPASS\tAT=>127178>127179>127180,>127178>8532789>127180;DP=13\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:13:8,5:-10.032681,-1.888646,-16.459288:39:-1.791860:15.570513:5\n",
      "1-chr1\t814303\t>127181>127183\tC\tG\t297.985\tPASS\tAT=>127181>127182>127183,>127181>8653557>127183;DP=13\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:13:0,13:-31.247666,-5.275909,-1.926448:13:-1.142152:18.333334:13\n",
      "1-chr1\t814309\t>127183>127185\tT\tG\t200.518\tPASS\tAT=>127183>127184>127185,>127183>8615453>127185;DP=21\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:21:10,11:-21.536960,-1.962264,-19.094007:171:-1.098612:18.333334:10\n",
      "1-chr1\t814378\t>127187>127189\tC\tT\t459.871\tPASS\tAT=>127187>127188>127189,>127187>8334973>127189;DP=24\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:24:2,22:-49.598588,-5.947414,-4.094601:18:-1.112549:22.521128:22\n",
      "1-chr1\t814380\t>127189>127191\tC\tG\t372.537\tPASS\tAT=>127189>127190>127191,>127189>8662192>127191;DP=22\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:22:3,19:-41.290872,-4.557627,-5.536146:19:-1.812108:22.521128:3\n",
      "grep: write error: Broken pipe\n"
     ]
    }
   ],
   "source": [
    "# we can see support for variants based on reads aligned from this sequencing run/sample\n",
    "!grep -P \"chr1\\t\" align.vcf | head -n 25"
   ]
  },
  {
   "cell_type": "markdown",
   "id": "244b7d8d-230c-4381-8f22-ba32e9a105fb",
   "metadata": {},
   "source": [
    "Now, these are variants we encoded into our graph. They could be related to an individual's ethnicity, disease state, etc.\n",
    "Following this, we usually want to call variants not included in our reference graph -- novel variants.\n",
    "\n",
    "The way variant calling in graphs works is to identify bubbles in the graph and establish read support for those bubbles. To\n",
    "call novel variants, we want to use alignments with discrepencies to our reference graph to add new bubbles to the graph.\n",
    "Then we treat this just like our above workflow for calling novel variants."
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "id": "ad9aa5b0-f410-4daa-a6a5-045fccc45cdc",
   "metadata": {},
   "outputs": [],
   "source": [
    "# We need to create a .vg/pg format from our autoindexed workflow for this workflow. It's annoying this isn't done for you \n",
    "# by autoindex currently. This is because no output from autoindex is acceptable by augment, and autoindex alters node ids from our\n",
    "# input gfa file, so the node ids from our initial gfa will not be the same as what is in the alignment.\n",
    "!vg convert index.xg > index.pg\n",
    "!vg augment index.pg align.sorted.gam -A aug.gam > augment.vg\n",
    "!vg index augment.vg -x augment.xg\n",
    "!vg pack -x augment.xg -g aug.gam -o aln.aug.pack\n",
    "# use -A in script, cehck\n",
    "!vg call augment.xg -k aln.aug.pack > align.aug.vcf"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 9,
   "id": "32404ca9-4b67-4217-93e9-d64cb1e8d902",
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
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      "1-chr1\t10234\t>9511549>13223538\tCTAACCCTAACCCTAAACCCTA\tTAACCCTAACCCTAACCCT,TAACCCTAACCCTACCCCT\t79.2035\tPASS\tAT=>9511549>9511550>9511551>9511552>9511553>9511554>9511555>9511556>9511557>9511558>9511559>9511560>9511561>9511562>9511563>9511564>9511565>9511566>9511567>9511568>9511569>9511570>9511571>9511572>102018>43603892>43603893>43603894>43603895>43603896>43603897>43603898>43603899>43603900>43603901>43603902>43603903>43603904>43603905>43603906>43603907>43603908>43603909>43603910>43603911>43603912>43603913>43603914>43603915>43603916>43603917>43603918>43603919>43603920>43603921>43603922>102019>13223521>13223522>13223523>13223524>13223525>13223526>13223527>13223528>13223529>13223530>13223531>13223532>13223533>13223534>13223535>13223536>13223537>13223538,>9511549>9511550>9511551>9511552>9511553>9511554>9511555>9511556>9511557>9511558>9511559>9511560>9511561>9511562>9511563>9511564>9511565>9511566>9511567>9511568>9511569>9511570>9511571>9511572>102018>43603892>43603893>43603894>43603895>43603896>43603897>43603898>43603899>43603900>43603901>43603902>43603903>43603904>43603905>43603906>43603907>43603908>43603909>43603910>43603911>43603912>43603914>43603915>43603916>43603917>43603918>43603919>43603920>43603921>43603922>102019>13223521>13223522>13223523>13223524>13223525>13223526>13223528>13223529>13223530>13223531>13223532>13223533>13223535>13223536>13223537>13223538,>9511549>9511550>9511551>9511552>9511553>9511554>9511555>9511556>9511557>9511558>9511559>9511560>9511561>9511562>9511563>9511564>9511565>9511566>9511567>9511568>9511569>9511570>9511571>9511572>102018>43603892>43603893>43603894>43603895>43603896>43603897>43603898>43603899>43603900>43603901>43603902>43603903>43603904>43603905>43603906>43603907>43603908>43603909>43603910>43603911>43603912>43603914>43603915>43603916>43603917>43603918>43603919>43603920>43603921>43603922>102019>13223521>13223522>13223523>13223524>13223525>13223526>13223528>48955101>13223530>13223531>13223532>13223533>13223535>13223536>13223537>13223538;DP=189\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/2:189:15,89,86:-1142.420327,-1137.884052,-1136.617595,-1136.584712,-1135.323218,-1138.263490:1:-7.790802:3320.773438:85\n",
      "1-chr1\t10327\t>39891997>39892014\tTAAC\tCAA\t463.578\tPASS\tAT=>39891997>39891998>39891999>39892000>39892001>39892002>39892003>39892004>39892005>39892006>39892007>39892008>39892009>39892010>39892011>39892012>39892013>39892014,>39891997>39891998>39891999>39892000>39892001>39892002>39892003>39892004>39892005>39892006>39892007>39892008>39892009>48955326>39892011>39892012>39892014;DP=148\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:148:109,39:-65.674810,-19.794028,-225.937608:18:-5.457260:242.919998:39\n",
      "1-chr1\t10352\t>8841611>8841628\tT\tTA\t930.959\tPASS\tAT=>8841611>8841612>8841613>8841614>8841615>8841616>8841617>8841618>8841619>8841620>8841621>8841622>8841623>8841624>8841625>8841626>8841627>8841628,>8841611>8841612>8841613>8841614>8841615>8841616>8841617>8841618>8841619>8841620>48955484>8841621>8841622>8841623>8841624>8841625>8841626>8841627>8841628;DP=147\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:147:87,60:-98.709119,-6.090126,-159.904409:197:-5.442418:187.240005:60\n",
      "1-chr1\t10463\t>42131989>102026\tA\tC\t65.6714\tPASS\tAT=>42131989>42131990>42131991>42131992>102026,>42131989>42131990>48955689>42131992>102026;DP=30\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:30:23,7:-13.000216,-6.910183,-50.480100:17:-2.096208:55.119999:6\n",
      "1-chr1\t10481\t>37240535>37240538\tAGCCG\tA\t246.139\tPASS\tAT=>37240535>37240536>37240537>37240538,>37240535>37240538;DP=18\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:18:5,13:-30.300828,-6.163988,-12.451663:59:-1.791761:41.232727:5\n",
      "1-chr1\t10517\t>28799058>28799060\tC\tT\t218.836\tPASS\tAT=>28799058>28799059>28799060,>28799058>48955845>28799060;DP=30\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:30:17,13:-23.617513,-2.210987,-32.650949:214:-1.098612:27.345455:13\n",
      "1-chr1\t10519\t>28799060>28799063\tG\tC\t496.68\tPASS\tAT=>28799060>28799061>28799062>28799063,>28799060>48955816>28799062>28799063;DP=29\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:29:4,25:-54.737715,-5.570181,-6.825712:19:-1.109656:27.345455:4\n",
      "1-chr1\t10532\t>102028>43806906\tAG\tGC\t516.862\tPASS\tAT=>102028>43806905>43806906,>102028>48955844>43806906;DP=30\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:30:4,26:-57.016838,-5.851476,-6.825712:9:-1.199464:27.345455:4\n",
      "1-chr1\t10560\t>43806918>102029\tC\tG\t108.542\tPASS\tAT=>43806918>43806919>102029,>43806918>48955856>102029;DP=37\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:37:28,9:-14.694951,-4.317844,-57.605904:103:-1.098612:35.720001:9\n",
      "1-chr1\t10568\t>32427422>32427424\tC\tG\t17.1328\tPASS\tAT=>32427422>32427423>32427424,>32427422>48955863>32427424;DP=37\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:37:32,5:-7.983485,-6.773299,-68.931192:12:-1.158421:35.720001:5\n",
      "1-chr1\t10581\t>32427430>32427433\tG\tA\t6.89808\tPASS\tAT=>32427430>32427431>32427432>32427433,>32427430>32427431>48955873>32427433;DP=34\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:34:30,4:-6.627562,-6.826931,-65.983603:9:-1.892154:35.720001:4\n",
      "1-chr1\t10611\t>20512461>20512463\tC\tG\t152.206\tPASS\tAT=>20512461>20512462>20512463,>20512461>48955843>20512463;DP=45\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:45:33,12:-19.258566,-4.515053,-67.477682:147:-1.098612:49.779999:12\n",
      "1-chr1\t10613\t>20512463>20512465\tA\tG\t132.228\tPASS\tAT=>20512463>20512464>20512465,>20512463>48955882>20512465;DP=44\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:44:33,11:-17.575406,-4.829720,-67.477682:146:-1.609438:49.779999:11\n",
      "1-chr1\t10621\t>20512468>20512471\tG\tC\t234.274\tPASS\tAT=>20512468>20512469>20512470>20512471,>20512468>20512469>48955883>20512471;DP=41\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:41:26,15:-26.143876,-3.193538,-50.837737:241:-1.098612:49.779999:15\n",
      "1-chr1\t10623\t>20512471>20512473\tT\tC\t938.895\tPASS\tAT=>20512471>20512472>20512473,>20512471>48955852>20512473;DP=41\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/1:41:0,41:-95.906800,-13.995872,-2.494258:115:-1.098612:49.779999:41\n",
      "1-chr1\t10634\t>25345461>25345465\tG\tC\t64.6407\tPASS\tAT=>25345461>25345462>25345463>25345464>25345465,>25345461>48955899>25345463>25345464>25345465;DP=54\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:54:44,10:-13.322730,-7.335767,-92.838771:6:-4.150148:49.779999:9\n",
      "1-chr1\t10661\t>40381283>40381296\tGCGCC\tCCACA\t200.187\tPASS\tAT=>40381283>40381284>40381285>40381286>40381287>40381288>40381289>40381290>40381291>40381292>40381293>40381294>40381295>40381296,>40381283>40381284>48955894>40381286>48955895>40381288>48955916>40381290>40381291>40381292>40381293>40381294>40381295>40381296;DP=122\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:122:98,24:-33.040845,-13.499207,-202.854256:11:-5.537501:135.279999:24\n",
      "1-chr1\t10690\t>102033>44667305\tGCGCC\tCCACA\t305.311\tPASS\tAT=>102033>44667298>44667299>44667300>44667301>44667302>44667303>44667304>44667305,>102033>48955918>44667299>48955919>44667301>48955920>44667303>44667304>44667305;DP=117\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:117:89,28:-40.540514,-10.486492,-180.689799:5:-5.609499:133.729996:28\n",
      "1-chr1\t10719\t>44667322>10075254\tGCGCC\tCCACA\t285.383\tPASS\tAT=>44667322>44667323>44667324>102034>10075249>10075250>10075251>10075252>10075253>10075254,>44667322>48955927>44667324>48955928>10075249>48955929>10075251>10075252>10075253>10075254;DP=116\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:116:89,27:-38.837776,-10.776520,-180.609024:16:-5.465205:132.179993:27\n",
      "1-chr1\t10748\t>10075268>10075271\tG\tC\t86.2938\tPASS\tAT=>10075268>10075269>10075270>10075271,>10075268>10075269>48955974>10075271;DP=114\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:114:96,18:-24.805729,-16.653449,-203.758064:39:-1.609542:90.153847:18\n",
      "1-chr1\t10750\t>10075271>35635557\tGCC\tACA\t100.075\tPASS\tAT=>10075271>10075272>10075273>10075274>102035>35635557,>10075271>48955975>10075273>48955976>102035>35635557;DP=94\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:94:78,16:-21.791255,-12.260905,-163.981862:25:-4.790414:90.153847:16\n",
      "1-chr1\t10815\t>41843380>41843382\tT\tTC\t96.8809\tPASS\tAT=>41843380>41843381>41843382,>41843380>41843381>48956040>41843382;DP=9\tGT:DP:AD:GL:GQ:GP:XD:MAD\t1/0:9:4,5:-11.026646,-1.815662,-8.471599:73:-1.098612:13.052631:4\n",
      "1-chr1\t10816\t>41843382>102037\tC\tCCA\t47.1612\tPASS\tAT=>41843382>102037,>41843382>48956032>102037;DP=10\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:10:7,3:-6.263340,-2.024360,-15.020568:48:-1.098627:13.052631:3\n",
      "1-chr1\t10821\t>102037>24109323\tT\tC\t6.83418\tPASS\tAT=>102037>24109322>24109323,>102037>48956035>24109323;DP=10\tGT:DP:AD:GL:GQ:GP:XD:MAD\t0/1:10:9,1:-2.887475,-3.103544,-20.271499:0:-1.698720:13.052631:1\n"
     ]
    }
   ],
   "source": [
    "# this file will contain both our known variants (above) as well as novel variants.\n",
    "!grep -P \"chr1\\t\" align.aug.vcf | head -n 25"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "id": "579b510d-68a8-4f6b-a407-e7efeef5657b",
   "metadata": {},
   "outputs": [],
   "source": []
  }
 ],
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