A quick walkthrough for PoPoolation. Starting with the reads the natural variation in a D. melanogaster may be assessed

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|| file || read number||read count|| read length|| || s_7_1_sequence_Jul2009.fastq||1||17957720||75|| || s_7_2_sequence_Jul2009.fastq||2||17957720||75||

NOTE: Data available at short read archiv http://trace.ddbj.nig.ac.jp/DRASearch/submission?acc=SRA023610

NOTE: A very small sample data set can alternatively be found in the Quick Guide: http://code.google.com/p/popoolation/wiki/TeachingPoPoolation

see [Manual]

PoPoolation may be obtained directly from the subversion repository. Just go to the directory where you want to install PoPoolation in the command line and enter the command:

svn checkout http://popoolation.googlecode.com/svn/trunk/ popoolation

To update your copy of PoPoolation with the latest improvements enter your PoPoolation directory and enter the command:

svn update

Alternatively PoPoolation may be downloaded from the project main page: http://code.google.com/p/popoolation/

However we recommend to use subversion as bugfixes will be immediately available in the repository

perl trim-fastq.pl --input1 s_7_1_sequence_Jul2009.fastq --input2 s_7_2_sequence_Jul2009.fastq --quality-threshold 20 --min-length 50 --output dmel_trimed

FINISHED: end statistics
Read-pairs processed: 17957720
Read-pairs trimmed in pairs: 13495995
Read-pairs trimmed as singles: 3802238


FIRST READ STATISTICS
First reads passing: 15656946
5p poly-N sequences trimmed: 16377
3p poly-N sequences trimmed: 61990
Reads discarded during 'remaining N filtering': 0
Reads discarded during length filtering: 2300774
Count sequences trimed during quality filtering: 8551460

Read length distribution first read
length  count
50  110528
51  112155
52  115546
53  119517
54  120622
55  123213
56  127909
57  132907
58  139580
59  143970
60  151528
61  166721
62  178000
63  185352
64  192283
65  202482
66  221528
67  247456
68  270715
69  312185
70  364457
71  461386
72  830395
73  1220619
74  9405892


SECOND READ STATISTICS
Second reads passing: 15137282
5p poly-N sequences trimmed: 99060
3p poly-N sequences trimmed: 35878
Reads discarded during 'remaining N filtering': 0
Reads discarded during length filtering: 2820438
Count sequences trimed during quality filtering: 9051269

Read length distribution second read
length  count
50  103225
51  102568
52  108397
53  117810
54  130272
55  146265
56  153847
57  166468
58  182299
59  191046
60  199036
61  192029
62  223088
63  224462
64  190667
65  183919
66  190513
67  202838
68  230031
69  269701
70  360601
71  460748
72  773657
73  1181002
74  8852793

First obtain a reference genome of D. melanogaster from http://flybase.org/

We used: dmel-all-chromosome-r5.22.fasta.gz

Remove everything after the first whitespace from the reference genome. This is a precautioniary measure as some mappers and software downstream of mapping have difficulties with fasta-ids containing whitespace.

awk '{print $1}' dmel-all-chromosome-r5.22.fasta > dmel-short-header.fa

Index the reference sequence

bwa index dmel-short-header.fa

Assuming the reference sequence is in the folder 'wg' the following command will map the reads which have been trimmed in a pair to the reference:

bwa aln -t 3 -o 2 -d 12 -e 12 -l 100 -n 0.01 wg/dmel-short-header.fa dmel_trimed_1 > dmel_trimed_1.sai
bwa aln -t 3 -o 2 -d 12 -e 12 -l 100 -n 0.01 wg/dmel-short-header.fa dmel_trimed_2 > dmel_trimed_2.sai

Converting mapping results to a sam file

bwa sampe wg/dmel-short-header.fa dmel_trimed_1.sai dmel_trimed_2.sai dmel_trimed_1 dmel_trimed_2 > dmel.sam

Filter by a mapping quality of 20 and convert the sam file into a sorted bam file. Filtering by a mapping qualiy of 20 removes the ambiguously mapped reads.

samtools view -q 20 -b -S dmel.sam|samtools sort - dmel.sort

samtools flagstat dmel.sort.bam

The result should be this

22467169 in total
0 QC failure
0 duplicates
22467156 mapped (100.00%)
22467169 paired in sequencing
11234128 read1
11233041 read2
22324803 properly paired (99.37%)
22439809 with itself and mate mapped
27347 singletons (0.12%)
73897 with mate mapped to a different chr
73897 with mate mapped to a different chr (mapQ>=5)

Convert the bam file into a pileup file:

samtools pileup dmel.sort.bam > dmel.pileup

Run the script Variance-sliding.pl with the dme.pileup file requesting Tajima's Pi. We use a window size and a step size of 10000.

perl Variance-sliding.pl --input dmel.pileup --output dmel.pi --measure pi --window-size 10000 --step-size 10000 --min-count 2 --min-coverage 4 --max-coverage 400 --min-qual 20 --pool-size 500

We assume a population size of 500 individuals. Furthermore we require a minimum allele count of 2, a minimum base quality of 20, a minimum coverage of 4 and a maximum coverage of 400.

Create the overview:

perl Visualise-output.pl --input dmel.pi --output dmel.pi.pdf --ylab pi --chromosomes "X 2L 2R 3L 3R 4"

and check out the result:

http://popoolation.googlecode.com/files/dmel.pi.pdf

The IGV may be downloaded from: http://www.broadinstitute.org/igv/

First convert the Drosophila melanogaster pi-file into a wiggle file

perl ~/dev/PopGenTools/VarSliding2Wiggle.pl --input dmel.pi --trackname "dmel Pi" --output dmel.pi.wig

than index the bam file:

samtools index dmel.sort.bam

Than:

1. open the IGV.
2. switch to the genome D. melanogaster reference 5.22
3. load the sorted bam file: dmel.sort.bam
4. load the wiggle file: dmel.pi.wig

and check out the results

http://popoolation.googlecode.com/files/igv_ex1.pdf

http://popoolation.googlecode.com/files/igv_ex2.pdf

Go to the FlyBase homepage (http://flybase.org/) and get the annotation: dmel-all-r5.22.gff.gz

Unzip the file.

Filter for exons and convert it into a gtf file:

cat dmel-all-r5.22.gff| awk '$2=="FlyBase" && $3=="exon"'| perl -pe 's/ID=([^:;]+)([^;]+)?;.*/gene_id "$1"; transcript_id "$1:1";/'> exons.gtf

Note: the script Variance-at-position.pl will group all exons with the same gene_id; the transcript_id is not considered

Note: UCSC (the Tables section) already provides gtf formated annotation -> if you use those and the proper reference sequence no reformating would be necessary

Run Variance-at-position.pl

perl Variance-at-position.pl --pool-size 500 --min-qual 20 --min-coverage 4 --min-count 2 --max-coverage 4 --pileup dmel.pileup --gtf exons.gtf --output dmel.genes.pi --measure pi

We set the pool size to 500, request pi as Population Genetic estimator, use the previously generated pileup file and the reformated exon annotation.

Output will be something like the following:

FBgn0042083 7   0.961   0.002524220
FBgn0027066 9   0.970   0.001810036
FBgn0033100 3   0.989   0.001972413
FBgn0033101 2   0.664   0.001424038
CG9438  8   0.900   0.003091130
FBgn0085421 14  0.936   0.003365280

• column 1: the gene id (or id of the region)
• column 2: the number of SNPs found in the gene
• column 3: covered fraction; how much of the gene is sufficiently covered by the pileup file; values 0-1; 0..not a single base of the gene has the required coverage, 1.. all bases of the gene have the required coverage
• column 4: the measure; here Tajima's pi

NOTE: This script is loading the whole annotation into the memory and internally converts it into a per-base hash. For very large gtf files this can be quite memory demanding. In case you do not have enough memory you may split your gtf file and analyse them separately. As long as you are not splitting genes, this will not affect the outcome. For example the following command will retrieve only entries from chromosome 2R;

cat exons.gtf| awk '$1=="2R"' > 2R-exons.gtf