Human Trios

Diploid genomes

The initial steps in calling variants for diploid or multi-ploid organisms with NGS data are the same as what we've already seen:

Expectations of output are quite different however, which can add statistical power to uncovering variation in populations or organisms with more than two expected variants at the same location. If you happen to be working with a model organism with extensive external data (ESPECIALLY HUMAN), then there are even more sophisticated tools like the Broad Institute's GATK that can improve both sensitivity and specificity of your variant calls.

Trio (or familial) analysis has been exceptionally powerful for identifying rare childhood diseases. The most prominent publication in this area is this first example of whole exome sequencing saving a life. There are many other publications since and some review articles such as this one. Familial analysis is critical for rare, autosomal dominant diseases because, almost by definition, the mutations may be "private" to each individual so we can't look across big populations to find one single causative mutation. But within families, we can identify bad private mutations in single genes or pathways and then look across populations to find commonality at the gene or pathway level to explain a phenotype.

Example: The CEU Trio from the 1000 Genomes Project

Many example datasets are available from the 1000 genomes project specifically for method evaluation and training. We'll explore a trio (mom, dad, child). Their accession numbers are NA12892, NA12891, and NA12878 respectively. To make the exercise run more quickly, we'll focus on data only from chromosome 20.

All the data we'll use is located here:

Diploid genome (human) example files

$BI/ngs_course/human_variation

This directory contains the following:

compressed raw data (the .fastq.gz files)
mapped data (the .bam files)
variant calls (the .vcf files)
the subdirectory ref with special references
.bam files containing a subset of mapped human whole exome data are also available on these three; those are the three files "NA*.bam".
We've pre-run samtools and GATK on each sample individually - those are the *GATK.vcf and *samtools.vcf files.
We've also pre-run samtools and GATK on the trio, resulting in GATK.all.vcf and samtools.all.vcf.
The 1000 Genomes project is really oriented to producing .vcf files; the file "ceu20.vcf" contains all the latest genotypes from this trio based on abundant data from the project.

Here we show the steps for:

Mapping
Single-sample variant calling with samtools
Multiple-sample variant calling with samtools

We'll return to this example data later to demonstrate a much more involved tool, GATK, to do the same steps in another tutorial.

Optional: Mapping Exercise (simplified way to map with bwa)

Expand here if you'd like to try this on your own...

Let's use Anna Battenhouse's shell script align_bwa.sh to align the paired end fastq file to the hg19 version of the genome calling the output trios_mapping. The input fastq file can be found at $BI/ngs_course/human_variation/allseqs_R1.fastq or if you have already copied today's materials to your scratch directory at $SCRATCH/BDIB_Human_tutorial/raw_files. Throughout the course we have been telling you to copy things to a local location before working with them, and you can continue to do so, but it is important to remember this is not necessarily a requirement.

If you use launcher_creator.py to create the job submission script. Be sure to append &> "meaningful file name" to the end of the script to capture the standard output and standard

Click here for a hint

Type align_bwa.sh without any options to gain insight into the programs expectations. As you might have put together, the "BWA" part of the script name represents the BWA mapping tool, but what isn't obvious is that it also uses SAMtools. Make sure that these things are available before starting the commands

If on an iDev node:

module load bwa
module load samtools
cds
cd BDIB_Human_tutorial
align_bwa.sh raw_files/allseqs_R1.fastq trios_mapping hg19 1

If you got an error message check here for a possible reason why

If your error message was this:

Fastq read1 File 'raw_files/allseqs_R1.fastq' not found...exiting

Did you remember to do this after the copy command finished?

cd $SCRATCH/BDIB_Human_tutorial/rawfiles
gunzip *.gz  # this will unzip all the compressed files you have just copied

Make job submission script for mapping & variant calling

echo "align_bwa.sh $BI/ngs_course/human_variation/allseqs_R1.fastq trios_mapping hg19 &> aln.trios_mapping.log" > commands  # this is an alternative way to generate a commands file than nano, use nano if more comfortable, or try this new option
launcher_creator.py -l map_call.sge -n map_call -t 01:00:00 -j commands -A UT-2015-05-18
module load bwa
module load samtools
qsub map_call.sge

"ls -lt" output ... (a way of checking if things completed correctly

-rw-rw-r-- 1 ded G-802740       392 May 28 03:13 trios_mapping.flagstat.txt
-rw-rw-r-- 1 ded G-802740   2149304 May 28 03:13 trios_mapping.sorted.bam.bai
-rw-rw-r-- 1 ded G-802740 334770401 May 28 03:13 trios_mapping.sorted.bam
-rw-rw-r-- 1 ded G-802740 411155732 May 28 03:12 trios_mapping.bam
-rw-rw-r-- 1 ded G-802740  62257040 May 28 03:01 trios_mapping.read2.sai
-rw-rw-r-- 1 ded G-802740  61884104 May 28 02:51 trios_mapping.read1.sai

"samtools flagstat trios_mapping.sorted.bam" expected results

4546280 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
3992266 + 0 mapped (87.81%:nan%)
4546280 + 0 paired in sequencing
2273140 + 0 read1
2273140 + 0 read2
40234 + 0 properly paired (0.88%:nan%)
3636928 + 0 with itself and mate mapped
355338 + 0 singletons (7.82%:nan%)
43366 + 0 with mate mapped to a different chr
15352 + 0 with mate mapped to a different chr (mapQ>=5)

Single-sample variant calling with samtools

We would normally use the BAM file from the previous mapping step to call variants in this raw data. However, for the purposes of this course we will use the actual BAM file provided by the 1000 Genomes Project (from which the .fastq file above was derived, leading to some oddities in it).

$SCRATCH/BDIB_Human_tutorial/raw_files/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam

With samtools, this is a two-step process:

samtools mpileup command transposes the mapped data in a sorted BAM file fully to genome-centric coordinates. It starts at the first base on the first chromosome for which there is coverage and prints out one line per base. Each line has information on every base observed in the raw data at that base position along with a lot of auxiliary information depending on which flags are set. It calculates the Bayseian prior probability given by the data, but does not estimate a real genotype.
bcftools with a few options added uses the prior probability distribution and the data to calculate a genotype for the variants detected.

Here are the commands, piped together (ONLY run this directly if you are in an idev session - NOT on a head node!):

Make sure you are on an idev node, or submit as job

The previous command will take quite a bit of time to complete. Therefore you may want to open a new terminal window, and log back into lonestar to do other things while the previous command continues to run.

Calling variants using samtools and bcftools

cds 
cd BDIB_Human_tutorial
mkdir samtools_example
cd samtools_example
samtools mpileup -uf ../raw_files/ref/hs37d5.fa ../raw_files/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam | bcftools view -vcg - > trios_tutorial.raw.vcf

submitted job option, showing command given at command line with the -b option

launcher_creator.py -n samtools_test -b "samtools mpileup -uf ref/hs37d5.fa NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam | bcftools view -vcg - > trios_tutorial.raw.vcf" -t 01:00:00 -q development -A UT-2015-05-18 -m samtools
qsub samtools_test.sge

Note that the reference chosen for mpileup must be exactly the same as the reference used to create the bam file. The 1000 genomes project has created it's own reference and so the command listed above won't work - we have to use the 1000 genomes reference which is $BI/ngs_course/human_variation/ref/hs37d5.fa or $SCRATCH/BDIB_Human_tutorial/raw_files/hs37d5.fa. We could have chosen another mapper if we'd wanted to though.

Exercise:

Write a modified version of the command above to use the proper reference into a commands file, create the job submission script with launcher_creator.py and submit the job.

Solution

As we did for mapping, we need to submit these to the Lonestar queue:

Submission of variant calling commands

echo "samtools mpileup -uf $SCRATCH/BDIB_Human_tutorial/raw_files/hs37d5.fa $SCRATCH/BDIB_Human_tutorial/raw_files/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam | bcftools view -vcg - > trios_tutorial.raw.vcf" > commands
launcher_creator.py -l call_vars.sge -n call_vars -t 01:00:00 -j commands -A UT-2015-05-18
qsub call_vars.sge

After your single-sample variant calling job completes

If you don't want to wait, CHANGE TO A NEW DIRECTORY and create a symbolic link to the completed file:

if on an iDEV node you will need to put the running command to the background first

mkdir single-sample-variant-speedup
cd single-sample-variant-speedup 
ln -s $SCRATCH/BDIB_Human_tutorial/raw_files/NA12878.chrom20.samtools.vcf trios_tutorial.raw.vcf  # this creates a new file called trios_tutorial.raw.vcf that has the same contents as the NA12878.chrom20.samtools.vcf file.

You can continue with the tutorial in this directory, but remember you can return to your job directory if you want to see your actual data.

You can examine some of the variant calls with:

more trios_tutorial.raw.vcf

Note the extensive header information, followed by an ordered list of variants.

A bcftools auxiliary perl script called vcfutils.pl can provide some useful stats. It's safe to run this on the head node since it's quick. This is not part of a standard TACC module but it's in our common $BI/bin directory.

Running vcfutils.pl

vcfutils.pl qstats trios_tutorial.raw.vcf

Expected output of vcfutils.pl script

login1$ vcfutils.pl qstats trios_tutorial.raw.vcf
QUAL #non-indel #SNPs #transitions #joint ts/tv #joint/#ref #joint/#non-indel
186	1011	1011	757	0	2.9803	0.0000	0.0000	2.9803
142	2036	2036	1441	0	2.4218	0.0000	0.0000	2.0059
122	3091	3091	2156	0	2.3059	0.0000	0.0000	2.1029
109	4177	4177	2925	0	2.3363	0.0000	0.0000	2.4259
99.5	5237	5237	3647	0	2.2937	0.0000	0.0000	2.1361
92	6273	6273	4354	0	2.2689	0.0000	0.0000	2.1489
85.5	7328	7328	5105	0	2.2964	0.0000	0.0000	2.4704
79	8352	8352	5811	0	2.2869	0.0000	0.0000	2.2201
74	9369	9369	6497	0	2.2622	0.0000	0.0000	2.0725
69	10553	10553	7311	0	2.2551	0.0000	0.0000	2.2000
65	11782	11782	8176	0	2.2673	0.0000	0.0000	2.3764
61	13059	13059	9090	0	2.2902	0.0000	0.0000	2.5179
57	14280	14280	9945	0	2.2941	0.0000	0.0000	2.3361
53	15301	15301	10656	0	2.2941	0.0000	0.0000	2.2935
48.8	16323	16323	11347	0	2.2803	0.0000	0.0000	2.0876
45	17460	17460	12122	0	2.2709	0.0000	0.0000	2.1409
41.8	18639	18639	12899	0	2.2472	0.0000	0.0000	1.9328
39.8	19660	19660	13572	0	2.2293	0.0000	0.0000	1.9339
37.8	21013	21013	14496	0	2.2243	0.0000	0.0000	2.1538
35.8	22309	22309	15380	0	2.2197	0.0000	0.0000	2.1456
33.8	23568	23568	16251	0	2.2210	0.0000	0.0000	2.2448
31.8	24846	24846	17101	0	2.2080	0.0000	0.0000	1.9860
29.8	26171	26171	17975	0	2.1931	0.0000	0.0000	1.9379
28	27308	27308	18688	0	2.1680	0.0000	0.0000	1.6816
26	28654	28654	19551	0	2.1478	0.0000	0.0000	1.7867
24	29947	29947	20371	0	2.1273	0.0000	0.0000	1.7336
22	31050	31050	21065	0	2.1097	0.0000	0.0000	1.6968
20	32081	32081	21719	0	2.0960	0.0000	0.0000	1.7347
17.1	33251	33251	22446	0	2.0774	0.0000	0.0000	1.6411
13.2	34447	34447	23238	0	2.0732	0.0000	0.0000	1.9604
10.4	35751	35751	24067	0	2.0598	0.0000	0.0000	1.7453
9.53	36772	36772	24724	0	2.0521	0.0000	0.0000	1.8049
8.65	38023	38023	25539	0	2.0457	0.0000	0.0000	1.8693
7.8	39301	39301	26360	0	2.0369	0.0000	0.0000	1.7965
6.98	40665	40665	27196	0	2.0192	0.0000	0.0000	1.5833
6.2	42394	42394	28243	0	1.9958	0.0000	0.0000	1.5352
5.46	44018	44018	29211	0	1.9728	0.0000	0.0000	1.4756
4.77	45553	45553	30168	0	1.9609	0.0000	0.0000	1.6557
4.13	47020	47020	31081	0	1.9500	0.0000	0.0000	1.6480
3.54	48572	48572	32063	0	1.9422	0.0000	0.0000	1.7228
3.01	50463	50463	33139	0	1.9129	0.0000	0.0000	1.3202

Here is an honest write-up about the Ts/Tv metric, referencing this 2002 paper by Ebersberger. Bottom line: between about 2.1 and 2.8 is OK for Ts/Tv (also called Ti/Tv). You can also get some quick stats with some linux one-liners on this page; there are more thorough analysis programs built to work with vcf's.

ADVANCED linux 1 liners to look at summary stats (indels and het vs. hom. alleles)

tacc:$SCRATCH/BDIB_Human_tutorial/single-sample-variant-speedup$ grep -c INDEL trios_tutorial.raw.vcf
3432
tacc:$SCRATCH/BDIB_Human_tutorial/single-sample-variant-speedup$ cat trios_tutorial.raw.vcf | awk 'BEGIN {FS=";"} {for (i=1;i<=NF;i++) {if (index($i,"AF1")!=0) {print $i} }}' | \
awk 'BEGIN {FS="="} {print int($2*10)/10}' | sort | uniq -c | sort -n -r | head
  36757 1
  19404 0.5
   1819 0.4
     36 0.6
     17 0.8
     16 0.7
      1 0

Keep in mind that variant files only record variation that can be seen with the data provided. Where ever sample sequence exactly matches the reference (i.e. is homozygous wildtype relative to the reference) there will be no data. Which looks the same as if you had no data in those regions; this leads us to our next topic.

Multiple-sample variant calling with samtools

This is all fine, but if you're actually trying to study human (or other organism) genetics, you must discriminate homozygous WT from a lack of data. This is done by providing many samples to the variant caller simultaneously. This concept extends further to populations; calling variants across a large and diverse population provides a stronger Bayesian prior probability distribution for more sensitive detection.

To instruct samtools to call variants across many samples, you must simply give it mapped data with each sample tagged separately. Samtools allows two methods to do this:

By providing separate bam files for each sample, like this:

samtools multi-sample variants: separate bam files

samtools mpileup -uf $SCRATCH/BDIB_Human_tutorial/raw_files/ref/hs37d5.fa \
  $SCRATCH/BDIB_Human_tutorial/raw_files/NA12878.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam \
  $SCRATCH/BDIB_Human_tutorial/raw_files/NA12891.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam \
  $SCRATCH/BDIB_Human_tutorial/raw_files/NA12892.chrom20.ILLUMINA.bwa.CEU.exome.20111114.bam \
    | bcftools view -vcg - > trios_tutorial.all.samtools.vcf

By providing one or more bam files, each containing mapped reads from multiple samples tagged with unique samtools @RG tags.
samtools multi-sample variants: one or more bam files using @RG
```
samtools mpileup -uf $SCRATCH/BDIB_Human_tutorial/raw_files/ref/hs37d5.fa all.bam | bcftools view -vcg - > trios_tutorial.all.raw.vcf
```

The output file from the first option is in $BI/ngs_course/human_variation as all.samtools.vcf

CAVEAT: If you intend to use the second option, you must make sure you tag your reads with the right RG tag; this can easily be done during the samse or sampe stage of mapping with bwa with the -r option, using samtools merge, with picard tools AddOrReplaceReadGroup command, or with your own perl/python/bash commands. Note that our align_bwa.sh script takes care of this detail for us.

Identify the lineage

If genetics works, you should be able to identify the child based strictly on the genotypes. Can you do it?

Hint...

You're trying to find the genotypes in the all.samtools.vcf and all.GATK.vcf files, and then use your knowledge of Mendelian inheritance to figure out which of the three samples is the only one that could be a child of the other two.

This linux one-liner should give you a snapshot of data sufficient to figure it out:

cat all.samtools.vcf | head -10000 | awk '{if ($6>500) {print $2"\t"$10"\t"$11"\t"$12}}' | grep "0/0" | sed s/':'/' '/g | awk '{print $2"\t"$5"\t"$8}' | tail -100 | sort | uniq -c

example output of sample solution

tacc:/scratch/01057/sphsmith/human_variation$ cat all.samtools.vcf | head -10000 | awk '{if ($6>500) {print $2"\t"$10"\t"$11"\t"$12}}' | grep "0/0" | sed s/':'/' '/g | awk '{print $2"\t"$5"\t"$8}' | tail -100 | sort | uniq -c 
     12 0/0	0/1	0/0
      5 0/0	0/1	0/1
      3 0/1	0/0	0/0
      4 0/1	0/0	0/1
      8 0/1	0/0	1/1
     43 0/1	0/1	0/0
     24 0/1	1/1	0/0
      1 1/1	0/1	0/0

Explanation of command, and discussion of the output

Here are the steps going into this command:

1) Dump the contents of all.samtools.vcf

2) Take the first 10,000 lines

3) If the variant quality score is greater than 500, then print fields 2 (SNP position), 10, 11, and 12 (the 3 genotypes).

4) Filter for only lines that have at least one homozygous SNP (exercise to the reader to understand why...)

5) Break the genotype call apart from other information about depth: "sed" turns the colons into spaces so that awk can just print the genotype fields.

6) Take the last 100 lines, sort them, then count the unique lines

Here is my interpretation of the data:

1) This method effectively looks at a very narrow genomic region, probably within a homologous recombination block.

2) The most telling data: the child will have heterozygous SNPs from two homozygous parents.

3) So all this data is consistent with column 1 (NA12878) being the child:

	 12 0/0	0/1	0/0
      5 0/0	0/1	0/1
      4 0/1	0/0	0/1
      8 0/1	0/0	1/1
     43 0/1	0/1	0/0
     24 0/1	1/1	0/0

"Outlier" data are:

      3 0/1	0/0	0/0

      1 1/1	0/1	0/0

This is, in fact, the correct assessment - NA12878 is the child.

GATK

GATK deserves it's own page which is here, but we've already run it and will now look at some differences in the SNP calls.

Look at the differences between single- and multiple-sample SNP calls, and between Samtools/Bcftools SNP calls and GATK SNP calls

To keep things a bit more separated use these commands to orient the relevant data files

cds
cd BDIB_Human_tutorial
mkdir method_comparison
cd method_comparison
cp ../raw_files/*.vcf .

Try to find your own way of answering each of the following and compare it to the results of these 1 liners.

A note on 1 liners

1 liners represent more advanced work, and it is unlikely that you will come up with the same method of using them without a large amount of time spent trying to do these types of things. They also include several linux commands not covered in this class, but answers may be possible in other ways that do not require these commands. These commands are listed here in a more advanced form to serve as a future reference if you are trying to answer these, or similar questions.

How many SNPs were called in each case?

for file in `ls *.vcf`; do echo "File: $file `cat $file | grep -v '^#' | wc -l`"; done

What's the overlap between all the single-sample SNP calls aggregated together and the multi-sample SNP calls?

cat NA128*samtools.vcf | grep -v '^#' | awk '{print $2}' | sort | uniq > all.single.samtools.snps
cat all.samtools.vcf | grep -v '^#' | awk '{print $2}' | sort | uniq > all.mutiple.samtools.snps
comm all.single.samtools.snps all.mutiple.samtools.snps | awk 'BEGIN {print "Only in single\tOnly in multiple\tIn both"; FS="\t"} {i[NF]++} END {print i[1]"\t"i[2]"\t"i[3]}'

In theory, GATK does a much better job ruling out false positives.

Are there some SNPs GATK calls with high confidence that Samtools doesn't call at all?

grep -v '^#' all.GATK.vcf | awk '{$2=$2"AAAAAAA"; print}' | sort -k 2 > g.v
grep -v '^#' all.samtools.vcf | awk '{$2=$2"AAAAAAA"; print}' | sort -k 2 > s.v
join -a 1 -1 2 -2 2 g.v s.v | awk '{if (NF==12) {print}}' | grep PASS > g.v.pass.only
cat g.v.pass.only | sort -k 6 -n -r | head

Some more in depth information about how the commands work

What's going on here:

1) Yank out all the variant calls (comments start with '#') and add a string of "AAAAAAA" to them to make "sort" do it's job in a way that "join" will later like

2) Join the two files using their chromosome position as the join field, but also include any lines from the GATK file that DON'T match the samtools file. Use "awk" to figure out which ones came only from GATK (they are missing a bunch of fields from the samtools variant calls), look only for those that GATK has labeled "PASS" and write them to a file.

3) Sort the resultant file on the variant quality value - take the top 10 lines.

You will note that many of these are complex variants, particularly insertions, so it's not too surprising that GATK does better. But here's a SNP that GATK does much better on:

chr21:34278313

It has an interesting quantitative signature though... you might want to look at it in IGV.

Other notes on bcftools

bcftools has many other sub-commands such as performing association tests and estimating allele frequency spectrums.

You can't get gene-context information from bcftools - you have to shift to variant annotation tools to do that.

Side-note on `samtools mpileup`

If you don't need a variant caller that uses a full Bayesian inference engine with robust filtering, protections, etc. but just want to look for weird base-artifacts within your mapped data, check out samtools mpileup output directly.