# Homework 5 (due Tuesday, November 12, at 11:59pm PST)¶

For solutions, see HW 5 - Solution Set.

## 1. Fitting¶

Do a linear fit to each of the Anscombe’s Quartet data sets, and plot the lines and data points. Here is some simple example code for doing a linear fit to x and y in Python:

x = [1,2,3,4]
y = [3,5,7,10] # 10, not 9, so the fit isn't perfect

fit = polyfit(x,y,1)
fit_fn = poly1d(fit) # fit_fn is now a function which takes in x and returns an estimate for y

plot(x,y, 'yo', x, fit_fn(x), '--k')


Submit as usual (done in an IPython Notebook, submitted via a gist; send me the nbviewer URL).

## 2. Variant calling¶

(Please do either this, or expression analysis, or both. You only need to do one.)

The below data is from one of Rich Lenski’s LTEE papers, the one on the evolution of citrate consumption in LTEE.

### Install software¶

You’ll want an m1.large or m1.xlarge for this.

First, we need to install the BWA aligner:

cd /root

tar xvfj bwa-0.7.5.tar.bz2
cd bwa-0.7.5a
make

cp bwa /usr/local/bin


We also need a new version of samtools:

cd /root
curl -O -L http://sourceforge.net/projects/samtools/files/samtools/0.1.19/samtools-0.1.19.tar.bz2
tar xvfj samtools-0.1.19.tar.bz2
cd samtools-0.1.19
make
cp samtools /usr/local/bin
cp bcftools/bcftools /usr/local/bin
cd misc/
cp *.pl maq2sam-long maq2sam-short md5fa md5sum-lite wgsim /usr/local/bin/


cd /mnt

curl -O http://athyra.idyll.org/~t/REL606.fa.gz
gunzip REL606.fa.gz

curl -O ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR098/SRR098042/SRR098042.fastq.gz


Note, this last URL is the “Fastq files (FTP)” link from the European Nucleotide Archive (ENA) for this sample: http://www.ebi.ac.uk/ena/data/view/SRR098042.

### Do the mapping¶

Now let’s map all of the reads to the reference. Start by indexing the reference genome:

cd /mnt

bwa index REL606.fa


Now, do the mapping of the raw reads to the reference genome:

bwa aln REL606.fa SRR098042.fastq.gz  > SRR098042.sai


Make a SAM file (this would be done with ‘sampe’ if these were paired-end reads):

bwa samse REL606.fa SRR098042.sai SRR098042.fastq.gz > SRR098042.sam


This file contains all of the information about where each read hits on the reference.

Next, index the reference genome with samtools:

samtools faidx REL606.fa


Convert the SAM into a BAM file:

samtools import REL606.fa.fai SRR098042.sam SRR098042.bam


Sort the BAM file:

samtools sort SRR098042.bam SRR098042.sorted


And index the sorted BAM file:

samtools index SRR098042.sorted.bam


At this point you can visualize with tview or Tablet.

‘samtools tview’ is a text interface that you use from the command line; run it like so:

samtools tview SRR098042.sorted.bam REL606.fa


The ‘.’s are places where the reads align perfectly in the forward direction, and the ‘,’s are places where the reads align perfectly in the reverse direction. Mismatches are indicated as A, T, C, G, etc.

You can scroll around using left and right arrows; to go to a specific coordinate, use ‘g’ and then type in the contig name and the position. For example, type ‘g’ and then ‘rel606:553093<ENTER>’ to go to position 553093 in the BAM file.

For the Tablet viewer, click on the link and get it installed on your local computer. Then, start it up as an application. To open your alignments in Tablet, you’ll need three files on your local computer: REL606.fa, SRR098042.sorted.bam, and SRR098042.sorted.bam.bai. You can copy them over using Dropbox, for example.

### Calling SNPs¶

You can use samtools to call SNPs like so:

samtools mpileup -uD -f REL606.fa SRR098042.sorted.bam | bcftools view -bvcg - > SRR098042.raw.bcf


(See the ‘mpileup’ docs here.)

Now convert the BCF into VCF:

bcftools view SRR098042.raw.bcf > SRR098042.vcf


### Examining SNPs¶

You can load the VCF file with this code in IPython notebook:

import csv

fp = open(filename, 'rb')
r = csv.reader(fp, delimiter='\t')

snp_calls = []
for n, row in enumerate(r):
if row[0].startswith('#'):
continue
chr, pos, _, ref, alt = row[:5]
pos = int(pos)

snp_calls.append((chr, pos, ref, alt))
return snp_calls


The full filename should be /mnt/SRR098042.vcf.

Look at a few of the locations in tview or Tablet. Do you believe the variant calls?

### Problems¶

1. Download the FASTQ.GZ file for SRR098038, and call SNPs.
2. Compare a few the SNPs in SRR098038 with the SNPs from SRR098042. Are the SNPs in one a subset or superset of the SNPs in the other?
3. SRR098038 is one of the Cit+ strains from 38,000 generations. Go find the location of the mutS mutation by finding the mutS gene in the genome of the reference strain here. Do you find the mutation? What is it, precisely? (Coordinates & ref -> variant.)

Hand in your two VCF files, together with your answer for #3.

## 3. RNAseq expression analysis¶

(Please do either this, or variant calling, or both. You only need to do one.)

Here we’re going to look at RNAseq differential expression. We’re going to be using data generated from running the Eel Pond mRNAseq protocol. You can feel free to run through all of this – it goes through a complete de novo assembly and quantitation of a bunch of mRNAseq data – but the whole thing takes about a week, so ... you might not want to. Below, I’ve given you access to some of the output files instead :).

### Install software¶

You’ll want an m1.large or m1.xlarge for this.

Start by installing R and some R packages:

apt-get -y install r-base-core r-cran-gplots


Next, install the EBSeq package for calculating differential expression, which comes as part of RSEM, the package we used in khmer-protocols for calculating expression levels:

cd /root
curl -O http://deweylab.biostat.wisc.edu/rsem/src/rsem-1.2.7.tar.gz
tar xzf rsem-1.2.7.tar.gz
cd rsem-1.2.7
make
cd EBSeq
make

echo 'export PATH=\$PATH:/root/rsem-1.2.7' >> ~/.bashrc
source ~/.bashrc


Create a working directory:

cd /mnt
mkdir diffexpr
cd diffexpr

curl -O http://athyra.idyll.org/~t/rsem-data.tar.gz
tar xvf rsem-data.tar.gz


This will result in four files, {1,2,6,7}.fq.genes.results. These are the first two 0 Hour and the first two 6 Hour time points from the Tulin et al. study on Nematostella development, run through the entire Eel Pond protocol (see khmer-protocols, above).

### Calculate differential expression¶

To do the differential expression calculation with EBSeq:

cd /mnt/diffexpr

rsem-generate-data-matrix {1,2,6,7}.fq.genes.results > genes.matrix
rsem-run-ebseq genes.matrix 2,2 genes.changed


Here, the ‘2,2’ means there are 2 conditions, each with two replicates.

The EBSeq output will be in ‘genes.changed’. Read the docs to understand what’s in the output file – you’re most interested in the PPDE (posterior probability that a transcript is differentially expressed) and the PostFC (posterior fold change) columns, columns 4 and 5.

### Problem: Load in data & plot¶

1. Please build an IPython Notebook that loads in the sample 1 and sample 6 data sets and plots the expression levels of genes in sample 1 on the X axis and sample 6 on the Y axis; below is some code taken from the in class notebook that you can adapt.

2. Load in the ‘genes.changed’ file (hint: you only need column 1) and plot the locations of genes that have changed.

A tip: instead of iterating over all the keys in the sample1 dictionary, you can iterate over just the keys in the dictionary created to hold the results of the ‘genes.changed’ file.

3. Grab the results in http://athyra.idyll.org/~t/rsem-data2.tar.gz – they are additional replicates (3.fq.genes.results and 8.fq.genes.results). Redo the differential expression analysis & plotting (note: use ‘3,3’ for rsem-run-ebseq).

A function to load in a genes.results file:

import csv

r = csv.DictReader(open(filename, 'rb'), delimiter='\t')
results = {}
for row in r:
gene = row['gene_id']
results[gene] = row

return results


Code to extract the expression values (FPKM) for two samples and put them in numpy array:

sample1 = load_results('1.fq.genes.results') # these are replicates

sample1v2 = []
for k in sample1:
s1 = float(sample1[k]['FPKM'])
s2 = float(sample2[k]['FPKM'])

if s1 == 0 or s2 == 0:
continue
else:
sample1v2.append((s1, s2))

sample1v2 = numpy.array(sample1v2)


Code to plot sample 1 vs sample 2:

x = plot(sample1v2[:,0], sample1v2[:,1], 'bo', alpha=0.1)
ax = axes()
ax.set_yscale('log')
ax.set_xscale('log')


Code to load in the ‘.changed’ file:

def load_changed(filename, ppee_threshold=.05):
d = {}
r = csv.reader(open(filename, 'rb'), delimiter='\t')
r.next()
for row in r:
gene_id, PPEE, PPDE, postfc, realfc = row
PPEE=float(PPEE)
PPDE=float(PPDE)
postfc = float(postfc)
realfc = float(realfc)
if PPEE > ppee_threshold: # skip those with probability of equal expression > 0.05
continue
d[gene_id] = (PPEE, PPDE, postfc, realfc)
return d