SnpEff & SnpSift

https://pcingola.github.io/SnpEff/
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SnpEff & SnpSift is a toolbox for genomic variant annotation, functional effect prediction, and...
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### Installing and running SnpEff on an Amazon EC2 instance

Source: https://pcingola.github.io/SnpEff/snpeff/running

This example demonstrates setting up SnpEff on a cloud instance, including downloading the latest core version, unzipping, installing a genome database, and dumping database information.

```bash
$ ssh -i ./aws_amazon/my_secret_key.pem ec2-user@ec2-54-234-14-244.compute-1.amazonaws.com

       __|  __|_  )
       _|  (     /   Amazon Linux AMI
      ___|\___|___|

[ec2-user@ip-10-2-202-163 ~]$ wget https://snpeff-public.s3.amazonaws.com/versions/snpEff_latest_core.zip
[ec2-user@ip-10-2-202-163 ~]$ unzip snpEff_latest_core.zip
[ec2-user@ip-10-2-202-163 ~]$ cd snpEff/
[ec2-user@ip-10-2-202-163 snpEff]$ java -jar snpEff.jar download -v hg19
00:00:00.000    Downloading database for 'hg19'
...
00:00:36.340    Done
[ec2-user@ip-10-2-202-163 snpEff]$ java -Xmx8g -jar snpEff.jar dump -v hg19 > /dev/null
00:00:00.000    Reading database for genome 'hg19' (this might take a while)
00:00:20.688    done
00:00:20.688    Building interval forest
00:00:33.110    Done.
```

--------------------------------

### SnpEff Configuration Example

Source: https://pcingola.github.io/SnpEff/download

Example of SnpEff configuration file, showing how to set the data directory for databases.

```properties
#---
# Databases are stored here
# E.g.: Information for 'hg19' is stored in data_dir/hg19/
#
# You can use tilde ('~') as first character to refer to your home directory.
# Also, a non-absolute path will be relative to config's file dir
#
#---
data.dir = ./data/

```

--------------------------------

### Install Antlr Library with Maven

Source: https://pcingola.github.io/SnpEff/download

Install the Antlr library JAR file into the local Maven repository.

```bash
# Go to 'lib' dir
cd SnpEff/lib

# Antlr
mvn install:install-file \
    -Dfile=antlr-4.5.1-complete.jar \
    -DgroupId=org.antlr \
    -DartifactId=antlr \
    -Dversion=4.5.1 \
    -Dpackaging=jar

```

--------------------------------

### Install BioJava Core Library with Maven

Source: https://pcingola.github.io/SnpEff/download

Install the BioJava core library JAR file into the local Maven repository.

```bash
# BioJava core
mvn install:install-file \
    -Dfile=biojava3-core-3.0.7.jar \
    -DgroupId=org.biojava \
    -DartifactId=biojava3-core \
    -Dversion=3.0.7 \
    -Dpackaging=jar

```

--------------------------------

### Download and Install SnpEff Core

Source: https://pcingola.github.io/SnpEff/snpeff/running

Download the latest SnpEff core program using wget or curl, then uncompress the ZIP file to install it on Unix or Mac systems.

```bash
# Download using wget
wget https://snpeff-public.s3.amazonaws.com/versions/snpEff_latest_core.zip

# If you prefer to use 'curl' instead of 'wget', you can type:
#     curl -L https://snpeff-public.s3.amazonaws.com/versions/snpEff_latest_core.zip > snpEff_latest_core.zip

# Install
unzip snpEff_latest_core.zip

```

--------------------------------

### Example SnpEff Command Line

Source: https://pcingola.github.io/SnpEff/help

This is an example of a full command line for running SnpEff, including memory allocation, jar file, reference genome, and input VCF file. This format is crucial when reporting issues.

```bash
java -Xmx8g -jar snpEff.jar GRCh37.75 sample.vcf ...
```

--------------------------------

### Install BioJava Structure Library with Maven

Source: https://pcingola.github.io/SnpEff/download

Install the BioJava structure library JAR file into the local Maven repository.

```bash
# BioJava structure
mvn install:install-file \
    -Dfile=biojava3-structure-3.0.7.jar \
    -DgroupId=org.biojava \
    -DartifactId=biojava3-structure \
    -Dversion=3.0.7 \
    -Dpackaging=jar

```

--------------------------------

### Example VCF Check Output

Source: https://pcingola.github.io/SnpEff/snpsift/vcfcheck

This example demonstrates the output of `vcfCheck` when encountering malformed VCF entries, including issues with INFO field headers and unsorted file errors.

```bash
$ java -jar SnpSift.jar vcfCheck bad.vcf

WARNING: Malformed VCF entryfile 'bad.vcf', line 7:
        Entry  : 3  148885779   .   A   ATT,AT  999.0   PASS    UK10KWES_AC=0,0;MDV=94
        Errors :
                INFO filed 'UK10KWES_AC' has 'Number=1' in header, but it contains '2' elements.
                Cannot find header for INFO field 'MDV'

WARNING: Malformed VCF entryfile 'bad.vcf', line 14:
        Entry  : 3  148890104   .   TCA T   .   .   .
        Errors :
                File is not sorted: Position '3:148890104' after position '3:148890105'
```

--------------------------------

### SnpEff Build Command Example

Source: https://pcingola.github.io/SnpEff/snpeff/build_db

This is the command used to initiate the SnpEff database build process for a specified genome.

```bash
java -jar snpEff.jar build -v genome_name
```

--------------------------------

### Example BED file output

Source: https://pcingola.github.io/SnpEff/snpeff/commands

Shows the beginning of a BED file generated by SnpEff dump. Note that BED coordinates are zero-based and semi-open.

```bash
$ head GRCh37.70.bed
1   0   249250621   Chromosome_1
1   111833483   111863188   Gene_ENSG00000134216
1   111853089   111863002   Transcript_ENST00000489524
1   111861741   111861861   Cds_CDS_1_111861742_111861861
1   111861948   111862090   Cds_CDS_1_111861949_111862090
1   111860607   111860731   Cds_CDS_1_111860608_111860731
1   111861114   111861300   Cds_CDS_1_111861115_111861300
1   111860305   111860427   Cds_CDS_1_111860306_111860427
1   111862834   111863002   Cds_CDS_1_111862835_111863002
1   111853089   111853114   Utr5prime_exon_1_111853090_111853114

```

--------------------------------

### Download PhastCons WigFix Files

Source: https://pcingola.github.io/SnpEff/snpsift/phastcons

Example command to download PhastCons wigFix files from UCSC for a specific genome (hg19).

```bash
mkdir -p ~/snpEff/db/phastCons && cd ~/snpEff/db/phastCons
wget -r -np -nd -A "*.phastCons100way.wigFix.gz" \
    http://hgdownload.soe.ucsc.edu/goldenPath/hg19/phastCons100way/hg19.100way.phastCons/
```

--------------------------------

### SnpSift Concordance Summary Example

Source: https://pcingola.github.io/SnpEff/snpsift/concordance

Example of a summary file generated by SnpSift concordance, showing sample counts and error types encountered during comparison.

```bash
$ cat concordance_genotype_sequencing.summary.txt
Number of samples:
    929    File genotype.vcf
    583    File sequencing.vcf
    514    Both files
Errors:
        ALT field does not match    19

```

--------------------------------

### SnpEff PDB Database Building Example

Source: https://pcingola.github.io/SnpEff/snpeff/build_pdb

An example command to build protein interaction data for a SnpEff database. It specifies the PDB directory and the ID mapping file, targeting the GRCh38.mane.1.0.ensembl genome version.

```bash
java -jar snpEff.jar pdb \
    -v \
    -pdbDir /path/to/pdb_files/ \
    -idMap /path/to/id_mapping.txt \
    GRCh38.mane.1.0.ensembl

```

--------------------------------

### Download Example Data for SnpEff Protocol

Source: https://pcingola.github.io/SnpEff/examples

Commands to download and unzip the sample data for the SnpEff protocol, including chromosome 7 and 17 data.

```bash
cd ~/snpEff
curl -v -L 'https://datasetsnpeff-public.s3.amazonaws.com/dataset/protocols.zip?sv=2019-10-10&st=2020-09-01T00%3A00%3A00Z&se=2050-09-01T00%3A00%3A00Z&si=prod&sr=c&sig=isafOa9tGnYBAvsXFUMDGMTbsG2z%2FShaihzp7JE5dHw%3D' > protocols.zip
unzip protocols.zip
```

--------------------------------

### GFF with FASTA Sequence Example

Source: https://pcingola.github.io/SnpEff/snpeff/build_db_gff_gtf

Demonstrates a GFF3 file structure that includes reference genome sequence after the '##FASTA' comment. This allows for a single file distribution.

```gff3
##gff-version 3
ctg123 . exon            1300  1500  .  +  .  ID=exon00001
ctg123 . exon            1050  1500  .  +  .  ID=exon00002
ctg123 . exon            3000  3902  .  +  .  ID=exon00003
ctg123 . exon            5000  5500  .  +  .  ID=exon00004
ctg123 . exon            7000  9000  .  +  .  ID=exon00005
##FASTA
>ctg123
cttctgggcgtacccgattctcggagaacttgccgcaccattccgccttg
tgttcattgctgcctgcatgttcattgtctacctcggctacgtgtggcta
tctttcctcggtgccctcgtgcacggagtcgagaaaccaaagaacaaaaa
aagaaattaaaatatttattttgctgtggtttttgatgtgtgttttttat
aatgatttttgatgtgaccaattgtacttttcctttaaatgaaatgtaat
cttaaatgtatttccgacgaattcgaggcctgaaaagtgtgacgccattc
...
```

--------------------------------

### SnpEff Regulatory Build Output Example

Source: https://pcingola.github.io/SnpEff/snpeff/build_reg

Example output from the SnpEff build process when creating regulatory databases from a GFF file. It shows progress and the saving of cell-type specific annotation files.

```text
Reading regulation elements (GFF)
    Chromosome '11' line: 226964
    Chromosome '12' line: 493780
    ...
    Chromosome '9'  line: 4832434
    Chromosome 'X'  line: 5054301
    Chromosome 'Y'  line: 5166958
Done
    Total lines                 : 5176289
    Total annotation count      : 3961432
    Percent                     : 76.5%
    Total annotated length      : 3648200193
    Number of cell/annotations  : 266
Saving database 'HeLa-S3' in file '/path/to/snpEff/data/GRCh37.65/regulation_HeLa-S3.bin'
Saving database 'HepG2' in file '/path/to/snpEff/data/GRCh37.65/regulation_HepG2.bin'
Saving database 'NHEK' in file '/path/to/snpEff/data/GRCh37.65/regulation_NHEK.bin'
Saving database 'GM12878' in file '/path/to/snpEff/data/GRCh37.65/regulation_GM12878.bin'
Saving database 'HUVEC' in file '/path/to/snpEff/data/GRCh37.65/regulation_HUVEC.bin'
Saving database 'H1ESC' in file '/path/to/snpEff/data/GRCh37.65/regulation_H1ESC.bin'
Saving database 'CD4' in file '/path/to/snpEff/data/GRCh37.65/regulation_CD4.bin'
Saving database 'GM06990' in file '/path/to/snpEff/data/GRCh37.65/regulation_GM06990.bin'
Saving database 'IMR90' in file '/path/to/snpEff/data/GRCh37.65/regulation_IMR90.bin'
Saving database 'K562' in file '/path/to/snpEff/data/GRCh37.65/regulation_K562.bin'
Done.
```

--------------------------------

### Example TXT file output

Source: https://pcingola.github.io/SnpEff/snpeff/commands

Shows the beginning of a tab-separated text file generated by SnpEff dump, containing gene, transcript, and exon information.

```bash
$ head GRCh37.70.txt
chr start       end        strand  type         id                          geneName  geneId            numberOfTranscripts  canonicalTranscriptLength  transcriptId     cdsLength  numerOfExons  exonRank  exonSpliceType
1   1           249250622  +1      Chromosome   1                                                                                                       
1   111833484   111863189  +1      Gene         ENSG00000134216             CHIA      ENSG00000134216   10                   1431                                                                  
1   111853090   111863003  +1      Transcript   ENST00000489524             CHIA      ENSG00000134216   10                   1431                       ENST00000489524  862        9                     
1   111861742   111861862  +1      Cds          CDS_1_111861742_111861861   CHIA      ENSG00000134216   10                   1431                       ENST00000489524  862        9                     
1   111861949   111862091  +1      Cds          CDS_1_111861949_111862090   CHIA      ENSG00000134216   10                   1431                       ENST00000489524  862        9                     
1   111853090   111853115  +1      Utr5prime    exon_1_111853090_111853114  CHIA      ENSG00000134216   10                   1431                       ENST00000489524  862        9             1         ALTTENATIVE_3SS
1   111854311   111854341  +1      Utr5prime    exon_1_111854311_111854340  CHIA      ENSG00000134216   10                   1431                       ENST00000489524  862        9             2         SKIPPED
1   111860608   111860732  +1      Exon         exon_1_111860608_111860731  CHIA      ENSG00000134216   10                   1431                       ENST00000489524  862        9             5         RETAINED
1   111853090   111853115  +1      Exon         exon_1_111853090_111853114  CHIA      ENSG00000134216   10                   1431                       ENST00000489524  862        9             1         ALTTENATIVE_3SS
1   111861742   111861862  +1      Exon         exon_1_111861742_111861861  CHIA      ENSG00000134216   10                   1431                       ENST00000489524  862        9             7         RETAINED

```

--------------------------------

### GTF File Format Example

Source: https://pcingola.github.io/SnpEff/snpeff/build_db_gff_gtf

This snippet shows a GTF file example that meets SnpEff's requirements, illustrating the definition of a gene, transcript, exons, start/stop codons, and UTR regions. Tabs are replaced by spaces for readability.

```gtf
# Note that tabs have been replaced by spaces for readability
chr1    ensembl  gene          10472288        10630758        .       +       .       gene_id "ENSG00000142655.13"; gene_type "protein_coding"; gene_name "PEX14";
chr1    ensembl  transcript    10474950        10630758        .       +       .       gene_id "ENSG00000142655.13"; transcript_id "ENST00000356607.9"; transcript_type "protein_coding";
chr1    ensembl  exon          10474950        10475002        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10474967        10475002        .       +       0       transcript_id "ENST00000356607.9";
chr1    ensembl  start_codon   10474967        10474969        .       +       0       transcript_id "ENST00000356607.9";
chr1    ensembl  exon          10495274        10495321        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10495274        10495321        .       +       0       transcript_id "ENST00000356607.9";
chr1    ensembl  exon          10536213        10536297        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10536213        10536297        .       +       0       transcript_id "ENST00000356607.9";
chr1    ensembl  exon          10599238        10599366        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10599238        10599366        .       +       2       transcript_id "ENST00000356607.9";
chr1    ensembl  exon          10618332        10618417        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10618332        10618417        .       +       2       transcript_id "ENST00000356607.9";
chr1    ensembl  exon          10623019        10623121        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10623019        10623121        .       +       0       transcript_id "ENST00000356607.9";
chr1    ensembl  exon          10624340        10624437        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10624340        10624437        .       +       2       transcript_id "ENST00000356607.9";
chr1    ensembl  exon          10627272        10627363        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10627272        10627363        .       +       0       transcript_id "ENST00000356607.9";
chr1    ensembl  exon          10629531        10630758        .       +       .       transcript_id "ENST00000356607.9";
chr1    ensembl  CDS           10629531        10629984        .       +       1       transcript_id "ENST00000356607.9";
chr1    ensembl  stop_codon    10629985        10629987        .       +       0       transcript_id "ENST00000356607.9";
chr1    ensembl  UTR           10474950        10474966        .       +       .       transcript_id "ENST00000356607.9"; 
chr1    ensembl  UTR           10629985        10630758        .       +       .       transcript_id "ENST00000356607.9"; 

```

--------------------------------

### Example GWAS Catalog Annotation and Output

Source: https://pcingola.github.io/SnpEff/snpsift/gwascatalog

Demonstrates running the GWAS Catalog annotation and then filtering the output VCF to show the added GWASCAT fields. This helps verify the annotation process.

```bash
$ java -jar SnpSift.jar gwasCat -genome GRCh37 -db gwascatalog.txt test.vcf > test.gwas.vcf

$ grep GWASCAT test.gwas.vcf | head -5

```

```text
1   1005806 rs3934834   C   T   .   PASS    AF=0.091;GWASCAT_TRAIT=Body_mass_index;GWASCAT_P_VALUE=1.0E-6;GWASCAT_REPORTED_GENE=AGRN;GWASCAT_PUBMED_ID=25673413
1   2069172 rs425277    C   T   .   PASS    AF=0.400;GWASCAT_TRAIT=Height;GWASCAT_P_VALUE=2.0E-8;GWASCAT_REPORTED_GENE=PRKCZ;GWASCAT_PUBMED_ID=20881960
1   2392648 rs2477686   G   C   .   PASS    AF=0.745;GWASCAT_TRAIT=Non_obstructive_azoospermia;GWASCAT_P_VALUE=3.0E-7;GWASCAT_REPORTED_GENE=MORN1;GWASCAT_PUBMED_ID=24532628
1   2513216 rs734999    C   T   .   PASS    AF=0.547;GWASCAT_TRAIT=Ulcerative_colitis;GWASCAT_P_VALUE=5.0E-13;GWASCAT_REPORTED_GENE=TNFRSF14;GWASCAT_PUBMED_ID=21297633
1   3083712 rs2651899   T   C   .   PASS    AF=0.467;GWASCAT_TRAIT=Migraine;GWASCAT_P_VALUE=1.0E-11;GWASCAT_REPORTED_GENE=PRDM16;GWASCAT_PUBMED_ID=23793025

```

--------------------------------

### Example FASTA CDS Headers

Source: https://pcingola.github.io/SnpEff/snpeff/build_db

Illustrates valid header formats for CDS FASTA files, showing transcript IDs and additional information.

```plaintext
>ENST00000448914
ACTGGGGGATACG

>chromosome:GRCh38:14:22449113:22449125:1 transcript:ENST00000448914.1 cds gene:ENSG00000228985.1 gene_biotype:TR_D_gene transcript_biotype:TR_D_gene gene_symbol:TRDD3 description:T cell receptor delta diversity 3
ACTGGGGGATACG

```

--------------------------------

### Example FASTA Protein Sequence Headers

Source: https://pcingola.github.io/SnpEff/snpeff/build_db

Illustrates valid header formats for protein sequences in a FASTA file, showing transcript IDs and associated gene/transcript information.

```fasta
>ENST00000382044
MPGEQMDPTGSQLDSDFSQQDTPCLIIEDSQPESQVLEDDSGSHFSMLSRHLPNLQTHKE
NPVLDVVSNPEQTAGEERGDGNSGFNEHLKENKVADPVDSSNLDTCGSISQVIEQLPQPN
RTSSVLGMSVESAPAVEEEKGEELEQKEKEKEEDTSGNTTHSLGAEDTASSQLGFGVLEL
...

>ENSP00000371475 pep chromosome:GRCh38:15:43403061:43493171:-1 gene:ENSG00000067369 transcript:ENST00000382044 gene_biotype:protein_coding transcript_biotype:protein_coding gene_symbol:TP53BP1 description:tumor protein p53 binding protein
MPGEQMDPTGSQLDSDFSQQDTPCLIIEDSQPESQVLEDDSGSHFSMLSRHLPNLQTHKE
NPVLDVVSNPEQTAGEERGDGNSGFNEHLKENKVADPVDSSNLDTCGSISQVIEQLPQPN
RTSSVLGMSVESAPAVEEEKGEELEQKEKEKEEDTSGNTTHSLGAEDTASSQLGFGVLEL
...
```

--------------------------------

### Uncompress Genotypes Example

Source: https://pcingola.github.io/SnpEff/snpsift/gt

Demonstrates how to decompress genotype information that was previously compressed by SnpSift GT, using the '-u' option to restore the original genotype fields.

```bash
$ java -jar SnpSift.jar gt -u test.gt.vcf
```

--------------------------------

### VCF Header PEDIGREE Information Example

Source: https://pcingola.github.io/SnpEff/snpeff/cansersamples

Illustrates the PEDIGREE meta-information format within a VCF header to define sample relationships. This is the default method.

```vcf
##PEDIGREE=<Derived=Patient_01_Somatic,Original=Patient_01_Germline>
#CHROM  POS ID  REF ALT QUAL    FILTER  INFO        FORMAT  Patient_01_Germline Patient_01_Somatic
1   69091   .   A   C,G .       PASS    AF=0.1122   GT      1/0                 2/1
1   69849   .   G   A,C .       PASS    AF=0.1122   GT      1/0                 2/1
1   69511   .   A   C,G .       PASS    AF=0.3580   GT      1/1                 2/2

```

--------------------------------

### Simplified Sequencing Data Analysis Pipeline

Source: https://pcingola.github.io/SnpEff/examples

A basic pipeline to process FASTQ sequencing data into an annotated VCF file. This is a highly simplified example and not a comprehensive tutorial.

```bash
# Download the genome, uncompress and rename file
wget ftp://ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r5.34_FB2011_02/fasta/dmel-all-chromosome-r5.34.fasta.gz
gunzip dmel-all-chromosome-r5.34.fasta.gz
mv dmel-all-chromosome-r5.34.fasta dm5.34.fasta

# Create a genome index (we assume you installed BWA)
bwa index -bwtsw dm5.34.fasta

# Map sequences to the genome: Create SAI file
bwa aln -bwtsw dm5.34.fasta s.fastq > s.sai

# Map sequences to the genome: Create SAM file
bwa samse dm5.34.fasta s.sai s.fastq > s.sam

# Create BAM file (we assume you installed SamTools)
samtools view -S -b s.sam > s.bam

# Sort BAM file (will create s_sort.bam)
samtools sort s.bam s_sort

# Create VCF file (BcfTools is part of samtools distribution)
samtools mpileup -uf dm5.34.fasta s_sort.bam | bcftools view -vcg - > s.vcf

# Analyze variants using snpEff
java -Xmx8g -jar snpEff.jar dm5.34 s.vcf > s.ann.vcf
```

--------------------------------

### GTF File Location Example

Source: https://pcingola.github.io/SnpEff/snpeff/build_db_gff_gtf

Specifies the expected directory structure for GTF files within the SnpEff data directory. Ensure the GENOME_NAME matches the snpEff.config file.

```bash
$SNPEFF_HOME/data/GENOME_NAME/genes.gtf
```

--------------------------------

### SnpEff Debug Log: Missing Start and Stop Codons

Source: https://pcingola.github.io/SnpEff/snpeff/refseq_mismatches

Example debug log messages from SnpEff indicating a transcript's CDS does not start with a valid start codon or end with a valid stop codon.

```log
WARNING: CDS for transcript 'NR_102370' does not start with a start codon:  TGG  TGGAAAAGACAATGATGTTTTATTTC...
WARNING: CDS for transcript 'NR_102370' does not end with a stop codon:     CCA  ...TCTGAGTTGTATGTGTGGACAGCACTGAGACTGAGTCTTTCCA
```

--------------------------------

### GFF File Location Example

Source: https://pcingola.github.io/SnpEff/snpeff/build_db_gff_gtf

Specifies the expected location for GFF files within the SnpEff installation directory. The genome name must match the configuration.

```bash
$SNPEFF_HOME/data/GENOME_NAME/genes.gff
```

--------------------------------

### Filter by Sample Name in SnpSift

Source: https://pcingola.github.io/SnpEff/snpsift/filter

Use sample names instead of numbers for more readable filter expressions, starting from version 4.1A. This example filters for somatic variants.

```bash
cat cancer.vcf | java -jar SnpSift.jar filter "GEN[Somatic].GT = '2/1'"
#CHROM  POS    ID   REF  ALT    QUAL  FILTER    INFO    FORMAT    Germline    Somatic
1       69091  .    A    C,G    .     PASS      AC=1    GT        1/0         2/1
```

--------------------------------

### List All Supported SnpEff Databases

Source: https://pcingola.github.io/SnpEff/snpeff/introduction

Run this command to list all available SnpEff databases. The output includes genome version, organism name, installation status, bundle name, and download link. Pipe the output to 'less' for easier navigation.

```bash
java -jar snpEff.jar databases | less

```

--------------------------------

### ANN Field Example

Source: https://pcingola.github.io/SnpEff/snpeff/inputoutput

An example of the ANN field in a VCF file, showing multiple annotations for a single variant.

```text
ANN=T|missense_variant|MODERATE|CCT8L2|ENSG00000198445|transcript|ENST00000359963|protein_coding|1/1|c.1406G>A|p.Gly469Glu|1666/2034|1406/1674|469/557||,
T|downstream_gene_variant|MODIFIER|FABP5P11|ENSG00000240122|transcript|ENST00000430910|processed_pseudogene||n.*397G>A|||||3944|
```

--------------------------------

### Build SnpEff from Source

Source: https://pcingola.github.io/SnpEff/download

Invoke the SnpEff build script after setting up necessary links.

```bash
cd $HOME/snpEff

# Create link to scripts_build directory if it doesn't exist
ln -s $HOME/workspace/SnpEff/scripts_build

# Invoke the build script
./scripts_build/make.sh

```

--------------------------------

### VCF Input Example

Source: https://pcingola.github.io/SnpEff/snpsift/rmrefgen

This is an example of a VCF file before processing with SnpSift RmRefGen. It shows sample genotypes, including homozygous reference (0/0).

```vcf
#CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	M1		M2		X1			X2
2L	426906	.	C	G	53.30	.	DP=169	GT:PL:GQ	0/1:7,0,255:4	0/1:7,0,255:4	0/0:0,0,0:6	0/0:0,30,255:35
2L	648611	.	A	T	999.00	.	DP=225	GT:PL:GQ	0/1:52,0,42:47	1/1:75,21,0:14	0/0:0,0,0:3	0/0:0,60,255:61
```

--------------------------------

### Download GTF and Reference Genome Files

Source: https://pcingola.github.io/SnpEff/snpeff/build_db

Commands to create a directory, download GTF annotation and reference genome FASTA files for a new genome build.

```bash
# Create directory for this new genome
cd /path/to/snpEff/data/
mkdir mm37.61
cd mm37.61

# Get annotation files
wget ftp://ftp.ensembl.org/pub/current/gtf/mus_musculus/Mus_musculus.NCBIM37.61.gtf.gz
mv Mus_musculus.NCBIM37.61.gtf.gz genes.gtf.gz

# Get the genome reference sequence file
cd /path/to/snpEff/data/genomes
wget ftp://ftp.ensembl.org/pub/current/fasta/mus_musculus/dna/Mus_musculus.NCBIM37.61.dna.toplevel.fa.gz
mv Mus_musculus.NCBIM37.61.dna.toplevel.fa.gz mm37.61.fa.gz
```

--------------------------------

### Example VCF Entry - GRCh37.63

Source: https://pcingola.github.io/SnpEff/snpeff/troubleshooting

This snippet shows an example VCF entry for transcript ENST00000487462 using GRCh37.63, where it is classified as 'protein_coding'. This is useful for comparing against newer database versions.

```vcf
1	protein_coding	exon	1655388	1655458	.	-	.	gene_id "ENSG00000008128"; transcript_id "ENST00000487462"; exon_number "1"; gene_name "CDK11A"; transcript_name "CDK11A-013";
1	protein_coding	exon	1653905	1654270	.	-	.	gene_id "ENSG00000008128"; transcript_id "ENST00000487462"; exon_number "2"; gene_name "CDK11A"; transcript_name "CDK11A-013";

```

--------------------------------

### Create Databases with SnpSift annotateMem

Source: https://pcingola.github.io/SnpEff/snpsift/annotate_mem

Use this command to create one or more databases from VCF files for later annotation. Specify the VCF files and the INFO fields to be used for database creation.

```bash
java -jar SnpSift.jar annmem \
  -create \
  -dbfile database_1.vcf -fields field_1,field_2,...,field_N \
  -dbfile database_2.vcf -fields field_1,field_2,...,field_N \
  ... \
  -dbfile database_N.vcf -fields field_1,field_2,...,field_N

```

--------------------------------

### List Available SnpEff Databases

Source: https://pcingola.github.io/SnpEff/download

List all available SnpEff databases using the SnpEff command.

```bash
java -jar snpEff.jar databases

```

--------------------------------

### Example Filtered VCF Output

Source: https://pcingola.github.io/SnpEff/snpsift/filter

This is an example of the VCF file content after applying the filter for missense variants in the TRMT2A gene. It shows the relevant VCF lines with ANN field details.

```vcf
22    20103915    .    C    T    .    .    ANN=T|stop_gained|HIGH|RANBP1|ENSG00000099901|transcript|ENST00000432879|protein_coding|1/3|c.208C>T|p.Arg70*|455/744|208/497|70/164||WARNING_TRANSCRIPT_INCOMPLETE
                                              ,T|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000439169|protein_coding|2/12|c.245G>A|p.Arg82His|561/2473|245/1932|82/643||
                                              ,T|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000252136|protein_coding|2/12|c.245G>A|p.Arg82His|634/2964|245/1878|82/625||
                                              ,T|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000403707|protein_coding|3/13|c.245G>A|p.Arg82His|607/2928|245/1878|82/625||
                                              ,T|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000404751|protein_coding|2/12|c.245G>A|p.Arg82His|584/2498|245/1689|82/562||
                                              ,T|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000445045|protein_coding|2/2|c.209G>A|p.Arg70His|432/582|209/359|70/118||WARNING_TRANSCRIPT_INCOMPLETE
                                              ,...

22    20103925    .    T    C    .    .    ANN=C|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000439169|protein_coding|2/12|c.235A>G|p.Asn79Asp|551/2473|235/1932|79/643||
                                              ,C|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000252136|protein_coding|2/12|c.235A>G|p.Asn79Asp|624/2964|235/1878|79/625||
                                              ,C|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000403707|protein_coding|3/13|c.235A>G|p.Asn79Asp|597/2928|235/1878|79/625||
                                              ,C|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000404751|protein_coding|2/12|c.235A>G|p.Asn79Asp|574/2498|235/1689|79/562||
                                              ,C|missense_variant|MODERATE|RANBP1|ENSG00000099901|transcript|ENST00000432879|protein_coding|1/3|c.218T>C|p.Phe73Ser|465/744|218/497|73/164||WARNING_TRANSCRIPT_INCOMPLETE
                                              ,C|missense_variant|MODERATE|TRMT2A|ENSG00000099899|transcript|ENST00000445045|protein_coding|2/2|c.199A>G|p.Asn67Asp|422/582|199/359|67/118||WARNING_TRANSCRIPT_INCOMPLETE
                                              ,C|splice_region_variant&intron_variant|LOW|RANBP1|ENSG00000099901|transcript|ENST00000430524|protein_coding|1/5|c.-374+7T>C||||||
                                              ,...

```

--------------------------------

### Annotated VCF Entry Example

Source: https://pcingola.github.io/SnpEff/snpsift/private

An example of a VCF entry annotated by SnpSift Private. The 'Private=Family_47' INFO field indicates that the variant is exclusively found in members of 'Family_47' as defined in the TFAM file.

```vcf
1   1005806 rs3934834   C   T   .   PASS    AF=0.091;Private=Family_47
```

--------------------------------

### Example LOF/NMD Tag Values

Source: https://pcingola.github.io/SnpEff/snpeff/inputoutput

This example shows how the LOF and NMD tags are populated based on variant effects. It illustrates the gene name, ID, total number of transcripts, and the percentage of affected transcripts.

```plaintext
LOF=(GAB4|ENSG00000215568|4|0.25);NMD=(GAB4|ENSG00000215568|4|0.25)

```

--------------------------------

### Running SnpEff using the wrapper script

Source: https://pcingola.github.io/SnpEff/snpeff/running

The wrapper script simplifies execution by automatically handling Java memory options and config file paths. This is the recommended way to run SnpEff for most users.

```bash
./scripts/snpEff GRCh37.75 examples/test.chr22.vcf > test.chr22.ann.vcf
```

--------------------------------

### Download and Prepare Annotation Files

Source: https://pcingola.github.io/SnpEff/snpeff/build_db

This snippet details the steps to download GTF, protein, CDS, and regulatory annotation files from Ensembl and prepare them for database construction. It also includes renaming files for SnpEff compatibility.

```bash
# Go to SnpEff's install dir
cd ~/snpeff

# Create database dir
mkdir data/GRCh37.70
cd data/GRCh37.70

# Download annotated genes
wget ftp://ftp.ensembl.org/pub/release-70/gtf/homo_sapiens/Homo_sapiens.GRCh37.70.gtf.gz
mv Homo_sapiens.GRCh37.70.gtf.gz genes.gtf.gz

# Download proteins
# This is used for:
#   - "Rare Amino Acid" annotations
#   - Sanity check (checking protein predicted from DNA sequences match 'real' proteins)
wget ftp://ftp.ensembl.org/pub/release-70/fasta/homo_sapiens/pep/Homo_sapiens.GRCh37.70.pep.all.fa.gz
mv Homo_sapiens.GRCh37.70.pep.all.fa.gz protein.fa.gz

# Download CDSs
# Note: This is used as "sanity check" (checking that CDSs predicted from gene sequences match 'real' CDSs)
wget ftp://ftp.ensembl.org/pub/release-70/fasta/homo_sapiens/cdna/Homo_sapiens.GRCh37.70.cdna.all.fa.gz
mv Homo_sapiens.GRCh37.70.cdna.all.fa.gz cds.fa.gz

# Download regulatory annotations
wget ftp://ftp.ensembl.org/pub/release-70/regulation/homo_sapiens/AnnotatedFeatures.gff.gz
mv AnnotatedFeatures.gff.gz regulation.gff.gz

# Uncompress
gunzip *.gz
```

--------------------------------

### Example FASTA Protein Sequence Output

Source: https://pcingola.github.io/SnpEff/snpeff/faq

The resulting FASTA file contains entries for reference and variant protein sequences, highlighting the changes introduced by a variant. Note the difference in the protein sequences.

```fasta
>ENST00000300658.9, gene: PGAP3, protein_id: ENSP00000300658.4, reference
MAGL....VAFAWVS...
>ENST00000300658.9, gene: PGAP3, protein_id: ENSP00000300658.4, variant: 17:39684602-39684602, ref:'C', alt:'T', HGVS.p: p.Ala143Thr
MAGL....VAFTWVS...
```

--------------------------------

### Example SnpSift Variant Type Output

Source: https://pcingola.github.io/SnpEff/snpsift/varianttype

This example shows the output of the SnpSift varType command, including variant type (SNP, INS, DEL, MNP) and zygosity (HOM, HET) flags in the INFO field.

```bash
$ java -jar SnpSift.jar varType test.vcf | grep -v "^#" | head
20  10469   .   C   G   100.0   PASS    SNP;HOM GT:AP   0|0:0.075,0.060
20  10492   .   C   T   100.0   PASS    SNP;HET GT:AP   0|1:0.180,0.345
20  10575   .   C   CG  100.0   PASS    INS;HET GT:AP   0|1:0.000,0.000
20  10611   .   CG  C   100.0   PASS    DEL;HET GT:AP   0|1:0.000,0.010
20  10618   .   GT  TA  100.0   PASS    MNP;HET GT:AP   0|1:0.020,0.030
```

--------------------------------

### SnpEff ANN Field Example for Cancer Analysis

Source: https://pcingola.github.io/SnpEff/snpeff/cansersamples

This example shows the ANN field output for a specific variant, illustrating multiple alternative alleles and their respective annotations, including somatic vs. germline comparisons.

```vcf
1   69511   .   A   C,G .   PASS    AF=0.3580;
                  ANN=C|missense_variant|MODERATE|OR4F5|ENSG00000186092|transcript|ENST00000335137|protein_coding|1/1|c.421A>C|p.Thr141Pro|421/918|421/918|141/305||
                     ,G|missense_variant|MODERATE|OR4F5|ENSG00000186092|transcript|ENST00000335137|protein_coding|1/1|c.421A>G|p.Thr141Ala|421/918|421/918|141/305||
                     ,G-C|missense_variant|MODERATE|OR4F5|ENSG00000186092|transcript|ENST00000335137|protein_coding|1/1|c.421C>G|p.Pro141Ala|421/918|421/918|141/305||
               GT  1/1 2/2
```

--------------------------------

### Download and Prepare dbNSFP Database Files

Source: https://pcingola.github.io/SnpEff/snpsift/dbnsfp

Instructions for developers to download, uncompress, and prepare dbNSFP database files for use with SnpSift. This involves creating a single tab-delimited file and compressing it with bgzip.

```bash
# Download dbNSFP database (adjust version as needed)
$ wget http://dbnsfp.houstonbioinformatics.org/dbNSFPzip/dbNSFP4.5c.zip

# Uncompress
$ unzip dbNSFP4.5c.zip

# Create a single file version
$ (head -n 1 dbNSFP4.5c_variant.chr1 ; cat dbNSFP4.5c_variant.chr* | grep -v "^#" ) > dbNSFP4.5c.txt

# Compress using block-gzip algorithm
bgzip dbNSFP4.5c.txt

```

--------------------------------

### Annotation with Putative Impact

Source: https://pcingola.github.io/SnpEff/snpeff/inputoutput

Example of the ANN field including the putative impact (HIGH, MODERATE, LOW, MODIFIER) of a variant.

```vcf
ANN=A|stop_gained|HIGH|||transcript|...
```
Tokens:
Raw