# Puzzles to help teach you more bedtools.

1. Create a BED file representing all of the intervals in the genome that are NOT exonic and are not Promoters (based on the promoters in the hESC file).

Answer:

    grep Promoter hesc.chromHmm.bed > hesc.promoters.bed

    cat exons.bed hesc.promoters.bed | sort -k1,1 -k2,2n | exons.and.promoters.bed

    bedtools complement -i exons.and.promoters.bed -g genome.txt > notexonsorpromoters.bed

2. What is the average distance from GWAS SNPs to the closest exon? (Hint - have a look at the closest tool.)

Answer:

    bedtools closest -a gwas.bed -b exons.bed -d | head
    chr1    1005805 1005806 rs3934834   chr1    1007125 1007955 NM_001205252_exon_0_0_chr1_1007126_r    0   -   1320
    chr1    1079197 1079198 rs11260603  chr1    1078118 1079434 NR_038869_exon_2_0_chr1_1078119_f   0   +   0
    chr1    1247493 1247494 rs12103 chr1    1247397 1247527 NM_001256456_exon_1_0_chr1_1247398_r    0   -   0
    chr1    1247493 1247494 rs12103 chr1    1247397 1247527 NM_001256460_exon_1_0_chr1_1247398_r    0   -   0
    chr1    1247493 1247494 rs12103 chr1    1247397 1247527 NM_001256462_exon_1_0_chr1_1247398_r    0   -   0
    chr1    1247493 1247494 rs12103 chr1    1247397 1247527 NM_001256463_exon_1_0_chr1_1247398_r    0   -   0
    chr1    1247493 1247494 rs12103 chr1    1247397 1247527 NM_017871_exon_1_0_chr1_1247398_r   0   -   0
    chr1    2069171 2069172 rs425277    chr1    2066700 2066786 NM_001033581_exon_1_0_chr1_2066701_f    0   +   2386
    chr1    2069171 2069172 rs425277    chr1    2066700 2066786 NM_001033582_exon_1_0_chr1_2066701_f    0   +   2386
    chr1    2069171 2069172 rs425277    chr1    2066700 2066786 NM_001242874_exon_1_0_chr1_2066701_f    0   +   2386

    bedtools closest -a gwas.bed -b exons.bed -d \
      | awk '{ sum += $11 } END { if (NR > 0) print sum / NR }'
    46713.1

3. Count how many exons occur in each 500kb interval (“window”) in the human genome. (Hint - have a look at the `makewindows` tool.)

Answer:

    bedtools makewindows -g genome.txt -w 500000 > genome.windows.bed
    bedtools intersect -a genome.windows.bed -b exons.bed -c > genome.windows.exoncount.bedg

or…

    bedtools makewindows -g genome.txt -w 500000 \
      | bedtools intersect -a - -b exons.bed -c \
    > genome.windows.exoncount.bedg

4. Are there any exons that are completely overlapped by an enhancer? If so, how many?

Answer:

    bedtools intersect -a exons.bed \
                       -b <(grep Enhancer hesc.chromHmm.bed) \
                       -wa -wb -f 1.0 \
    | head
    chr1    948846  948956  NM_005101_exon_0_0_chr1_948847_f    0   +   chr1    948337  949337  4_Strong_Enhancer
    chr1    1051439 1051736 NM_017891_exon_9_0_chr1_1051440_r   0   -   chr1    1051337 1051737 6_Weak_Enhancer
    chr1    1109285 1109306 NM_001130045_exon_0_0_chr1_1109286_f    0   +   chr1    1108537 1109537 6_Weak_Enhancer
    chr1    1109803 1109869 NM_001130045_exon_2_0_chr1_1109804_f    0   +   chr1    1109737 1109937 6_Weak_Enhancer
    chr1    1219357 1219470 NM_001130413_exon_4_0_chr1_1219358_f    0   +   chr1    1219137 1220137 7_Weak_Enhancer
    chr1    1219357 1219470 NR_037668_exon_4_0_chr1_1219358_f   0   +   chr1    1219137 1220137 7_Weak_Enhancer
    chr1    1229202 1229313 NM_030649_exon_1_0_chr1_1229203_r   0   -   chr1    1228937 1229937 6_Weak_Enhancer
    chr1    1229469 1229579 NM_030649_exon_2_0_chr1_1229470_r   0   -   chr1    1228937 1229937 6_Weak_Enhancer
    chr1    1234724 1234736 NM_030649_exon_14_0_chr1_1234725_r  0   -   chr1    1234137 1234937 7_Weak_Enhancer
    chr1    1245060 1245231 NM_153339_exon_4_0_chr1_1245061_f   0   +   chr1    1244937 1245337 4_Strong_Enhancer

    bedtools intersect -a exons.bed \
                       -b <(grep Enhancer hesc.chromHmm.bed) \
                       -wa -wb -f 1.0 -u \
    | wc -l
    13746

5. What fraction of the GWAS SNPs are exonic?

Answer (Any idea why we need -u?):

    wc -l gwas.bed
    17680 gwas.bed

    bedtools intersect -a gwas.bed -b exons.bed -u | wc -l
    1625

    echo "foo" | awk '{print 1625/17680}'
    0.0919118

6. What fraction of the GWAS SNPs are lie in either enhancers or promoters in the hESC data we have?

Answer (Any idea why we need -u?):

    bedtools intersect -a gwas.bed -b <(egrep "Enhancer|Promoter" hesc.chromHmm.bed) -u \
    | wc -l
    1285

    echo "foo" | awk '{print 1285/17680}'
    0.072681

7. Create intervals representing the canonical 2bp splice sites on either side of each exon (don’t worry about excluding splice sites at the first or last exon). (Hint - have a look at the flank tool.)

Answer:

    bedtools flank -l 2 -r 2 -i exons.bed -g genome.txt > splice-sites.bed

Or:

    bedtools slop -b 2 -i exons.bed -g genome.txt > exons.plus2.bed
    bedtools subtract -a exons.plus2.bed -b exons.bed > splice-sites.bed

8. What is the Jaccard statistic between CpG and hESC enhancers? Compare that to the Jaccard statistic between CpG and hESC promoters. Does the result make sense? (Hint - you will need `grep`).

Answer:

    bedtools jaccard -a cpg.bed -b <(grep Enhancer hesc.chromHmm.bed)
    intersection    union-intersection  jaccard n_intersections
    1148180 132977386   0.0086344   4969

    bedtools jaccard -a cpg.bed -b <(grep Promoter hesc.chromHmm.bed)
    intersection    union-intersection  jaccard n_intersections
    15661111    53551816    0.292448    20402

9. What would you expect the Jaccard statistic to look like if promoters were randomly distributed throughout the genome? (Hint - you will need the shuffle tool.)

Answer:

    bedtools shuffle -i <(grep Promoter hesc.chromHmm.bed) -g genome.txt \
      | sort -k1,1 -k2,2n \
    > promoters.shuffled.bed

    bedtools jaccard -a cpg.bed -b promoters.shuffled.bed
    intersection    union-intersection  jaccard n_intersections
    294071  68556207    0.00428949  78

10. Which hESC ChromHMM state (e.g., 11_Weak_Txn, 10_Txn_Elongation) represents the most number of base pairs in the genome? (Hint: you will need to use `awk` or `perl` here, as well as the groupby tool.)