shenlab-sinai / ngsplot

Quick mining and visualization of NGS data by integrating genomic databases
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I keep getting flat metaplots from the ngsplot? Genome loading issue or data issue? #39

Open GoogleCodeExporter opened 9 years ago

GoogleCodeExporter commented 9 years ago
What steps will reproduce the problem?

1. downloaded and installed the arabidopsis genome files 

ngsplotdb.py install ngsplot_Tair10_....gz

genome installed

2. run ngs.plot.r

ngs.plot.r -G Tair10 -R genebody -C /path/to/sorted_indexed/bamfile.bam -O 
output 

3.

What is the expected output? What do you see instead?

The expected output is any kind of non-flat metaplot! I get a flat unchanging 
metaplot across any region I choose genebody, tss tes etc.... but I don't get 
any errors that I can tell.

What version of the product are you using? On what operating system?

I've tried the most recent ngsplot and several of the older ones back to 2.00. 
I'm using a linux ubuntu 14.04 OS 

Please provide any additional information below.

it seems when I run the program I get large counts from the count files but 
both the plot files: heatmap and profile for genebody, tss, tes and anything 
else I try are completely flat as though no data has mapped there! I feel like 
its probably a genome issue but I don't know what I've done wrong in loading 
it! Any advice on what to check out?

Best Regards
DS

Original issue reported on code.google.com by sandman2...@gmail.com on 18 Feb 2015 at 3:50

GoogleCodeExporter commented 9 years ago
I've now gotten further see below 

Dear Shen Lab,
I wanted to first thank you all for all your hard work on such a useful tool! 
Unfortunately in my hands I cannot get it give me anything but flat plots.

I believe all genomes and files and dependencies are correct and I run:

ngs.plot.r -G Tair10 -R genebody -C my_sorted_indexed.bam -O output

It reports a flat plot an empty heatmap, no errors....
I found this post by Dr. Toshiyuki Sone where he was able to get his plots 
working by altering the bam files:

https://groups.google.com/forum/#!searchin/ngsplot-discuss/flat$20plot/ngsplot-d
iscuss/mWxJIYRlz_w/tcnQY2mGXkcJ

I believe this may be the same issue for me in that the: .Rdata files have 
lowercase  chr1, chr2 ....

and my bam files have Chr1 Chr2 .... I've tried many methods but cannot get 
ngs.plot.r to take my altered bam files

it mostly reports: Error: Read XZ5_AAG_edited.bam error: Inconsistent 
chromosome names in bam file. Check bam header.

Although I am fairly sure that the bam files are consistent. is there some way 
to alter either bam files or the .Rdata files that will allow me to profile my 
reads?

Best
DS

Original comment by sandman2...@gmail.com on 20 Feb 2015 at 7:49

GoogleCodeExporter commented 9 years ago
Figured it out! Thanks for such an amazing program. I had to remake my index 
files to contain small chr1,chr2 instead of large Chr1, Chr2 ... because the 
small chr1 .. are the ones that are read by your Rdata files. So far the 
results look very promising for our work. Thanks for all your help.

Best
DS 

Original comment by sandman2...@gmail.com on 23 Feb 2015 at 11:10

orzechoj commented 9 years ago

Hi Shen Lab,

I have a similar problem: Running ngs.plot.r produces plots where all values are 0. I run the following command

ngs.plot.r -G mm10 -R genebody -C my.bam -O outPrefix

(I installed the mm10 genome, and I didn't get any problems there.) When I run the program on the supplied example

ngs.plot.r -G hg19 -R tss -C hesc.H3k4me3.1M.bam -O hesc.H3k4me3.tss -T H3K4me3 -L 3000 -FL 300

everything worked ok.

I checked that the chromosome names in the mm10 genome.coord object are the same as in the bam file, e.g. "chr12", so for me that's not the problem. I also tried "-R tss", and/or "-D refseq" and got similar results. Also, "-MQ 1", "-F rnaseq" and "-SS same" didn't change anything.

(I ran this using ngsplot-2.47 as well as the ngsplot-develop version downloaded from gitHub 4/26-2015.)

Do you have any ideas what I could try next?

kind regards /Jakub

lishen commented 9 years ago

I'm not sure what is going on. Can you show us your bam's head?

Li

On Mon, Apr 27, 2015 at 10:43 AM, orzechoj notifications@github.com wrote:

Hi Shen Lab,

I have a similar problem: Running ngs.plot.r produces plots where all values are 0. I run the following command

ngs.plot.r -G mm10 -R genebody -C my.bam -O outPrefix

(I installed the mm10 genome, and I didn't get any problems there.) When I run the program on the supplied example

ngs.plot.r -G hg19 -R tss -C hesc.H3k4me3.1M.bam -O hesc.H3k4me3.tss -T H3K4me3 -L 3000 -FL 300

everything worked ok.

I checked that the chromosome names in the mm10 genome.coord object are the same as in the bam file, e.g. "chr12", so for me that's not the problem. I also tried "-R tss", and/or "-D refseq" and got similar results. Also, "-MQ 1", "-F rnaseq" and "-SS same" didn't change anything.

(I ran this using ngsplot-2.47 as well as the ngsplot-develop version downloaded from gitHub 4/26-2015.)

Do you have any ideas what I could try next?

kind regards /Jakub

— Reply to this email directly or view it on GitHub https://github.com/shenlab-sinai/ngsplot/issues/39#issuecomment-96681168 .

Li Shen, Ph.D. Computer Science Assistant Professor Department of Neuroscience Icahn School of Medicine at Mount Sinai New York, NY 10029 ################# ngs.plot - the coolest NGS plotting tool: https://code.google.com/p/ngsplot/ Want ChIP-seq differential analysis? Use: https://code.google.com/p/diffreps/

orzechoj commented 9 years ago

Hi Li, thank you for the quick reply,

The bam file I'm using is a bit weird. The library was generated using a special protocol, so what I have is paired end data, but only the second read in each pair is used. The data were mapped with bowtie2. Below are the first 10 lines, apologies for the lousy formatting.

best, Jakub

$ samtools view R1_formated_mapped_filtered_annotated_SMALL_SORT.bam | head -n 10

NS500688:7:H5FYJBGXX:2:13110:2209:5781 153 chr10 3139861 0 76M1I37M = 3139861 0 ATATTTGGAGATAAATTAGTTGGTCCATAGAGTCTTATCTGGGGAAGACAGGTCCGATAAGCCTCTACAATGCCTTGGATTCCATTGCAATAGGGGTTCGATGGGTTAAAGCTG 7FA)FFFFAAF)F.F7))<)<.7)FF..AFF<)FF<FAA7FFFFFFAF.FFFFA<FF7F<FFA<F<FAFFFFFFFF<FFFFFFFFFFFFFFFFFFF.AFFFFFFF7FFFFFFFF AS:i:-53 XN:i:0 XM:i:11 XO:i:1 XG:i:1 NM:i:12 MD:Z:3A7A10A4C13A12T12T32G9T0C0C0 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:22103:17410:17927 153 chr10 3140296 8 5M1I90M = 3140296 0 GTCTCTTTTTTTTCATTTATGCATTCATATTCAACAGGTGAGCTTCTGGAGGCTTCTTTGAGTTTTGTCATGGTGGTTTGAAATGTTCCAATGAGA FFFF<FFFFFFFFAFFAFFFFFFAFFFFFFFFAFFFFA<FFFF<.FFFFFFFF)7FFFFFFF.AFFF<FFFFF.FF))FFFFFFFAFFFFFFFAFF AS:i:-33 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:24A9T1A4T40A12 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22510:19317:1639 153 chr10 3140491 3 23M1D93M = 3140491 0 GCTTCCACATGGGATTCAGGTTGTTTTTTATAACCTCTGTTCGGTGAACCATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGA FA<<FF7<F7FFFF).FFFF7FF<F7AFAFFFAFFAFFFAAFFFAFFFFFFFFFF<<FFAFFFFFFFFFFFFFFFFF<FF<FFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFF AS:i:-42 XN:i:0 XM:i:7 XO:i:1 XG:i:1 NM:i:8 MD:Z:6C1G14^T11T0C6A20T0C51 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:13206:16212:9184 153 chr10 3140541 24 116M = 3140541 0 CATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGT FF<FFFFFFF7F<FFFFFFFFFFAFFFF<<FFFFF<FF<FFFFFFFF<FFFFFFFAFFFFFAFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-25 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:14T0C77T1C4G15 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:13504:14353:9068 153 chr10 3140544 23 116M = 3140544 0 CAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGTTAT FFFFAFF..FFFF.AFFF.F.F.FFAAF.FFFFFFFFFA<7A7)AF)FFFFF<FFFF7A<FFFFAFFFFFAFFFFFFF..F7F<FFFAAFFFA.FFFAAFFAFFFFFF.F<F7AFF AS:i:-30 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:11T0C77T1C4G16G1 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:23503:14378:11418 153 chr10 3140637 0 115M = 3140637 0 TTCAAACAATACCACTCTGTTATCCTTTATTTCTTCTGCTGAAATCGTAATACTCCCTTTCCCTGCAGGTTTGCCATTTTTGAGCACCAGTGGTCGAGTTAGCTTCTTACTGGAA AFFFA7FF<<FFFFFFF7FFFFAFFAF.FFFFFAFF7FFFFFFFFAFAF<FFFAF.FFAFFFFFFFFFFF.FFFFFFF<7FFFFFAF7FFFFFAAFFFFFFFAFFFFFFFFFFFA AS:i:-65 XN:i:0 XM:i:13 XO:i:0 XG:i:0 NM:i:13 MD:Z:4G16G15A7T1C3G6C6A26A1T0A5T10T2 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:23310:16699:2534 153 chr10 3361500 42 115M = 3361500 0 TTTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA FFAAAF7F7<FFA.FFF<FF.F<.7FFFFFFFF.FFFFAFFFFAAFFFFFFFFFFFFFFFAFFAFFFFFFAFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:11207:15928:8706 153 chr10 3361501 42 114M = 3361501 0 TTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA AAFAFAFFFFFFAFFAFFFAAFFF<F.FFFFF.FFFFFFFFFF<FFFFFFFFFFAFFFFFFFFFFFFFFFFFF7FFFFFF<FFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:12108:17217:19287 137 chr10 3367730 42 89M = 3367730 0 TACACACATACACATGATGGAGGGAGAAAAATACCCAAAGCCAAATGGTTTCCCGAGAGTCTTAAGAAACAAGGGTTCTCAATGAAGTG <AF.F.FFF<FFA7FFAAFFFFFFFF<<.A.F.))F...FFF)).FFFA<A.AFF.F<<AF.A.7<...A..FAF)7AFA<7.F.<A7< AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:33A55 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:12601:5782:3269 137 chr10 3381708 42 111M = 3381708 0 TTTATTTTGTATAATGACGTGATCCTAAAGTATCATTAAGTAGAATCACTCAACCAAGTGCCACAAAACTTTGGGATATAGCTAGTTACCCCATTTTTAAAACGAGAAAGC FFA)F7<.)AFA.FFA<F.)AFAFF.7A7FFFFF<FF.FFA7FFFFFFAFFF<FF.<FFFFF.F.A.)FF<FA)FF<7AA.AF.FFAF<)AFA7FFAA7.F)F<FA<FFFF AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:19G91 YT:Z:UP XF:Z:__no_feature

On Tue, Apr 28, 2015 at 9:00 AM, Jakub Westholm jakub.westholm@gmail.com wrote:

Hi Li, thank you for the quick reply,

The bam file I'm using is a bit weird. The library was generated using a special protocol, so what I have is paired end data, but only the second read in the pair is used. The data were mapped with bowtie2. Below are the first 50 lines, apologies for the lousy formatting.

best, Jakub

$ samtools view my.bam | head -n 50

NS500688:7:H5FYJBGXX:2:13110:2209:5781 153 chr10 3139861 0 76M1I37M = 3139861 0 ATATTTGGAGATAAATTAGTTGGTCCATAGAGTCTTATCTGGGGAAGACAGGTCCGATAAGCCTCTACAATGCCTTGGATTCCATTGCAATAGGGGTTCGATGGGTTAAAGCTG 7FA)FFFFAAF)F.F7))<)<.7)FF..AFF<)FF<FAA7FFFFFFAF.FFFFA<FF7F<FFA<F<FAFFFFFFFF<FFFFFFFFFFFFFFFFFFF.AFFFFFFF7FFFFFFFF AS:i:-53 XN:i:0 XM:i:11XO:i:1 XG:i:1 NM:i:12 MD:Z:3A7A10A4C13A12T12T32G9T0C0C0 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:22103:17410:17927 153 chr10 3140296 8 5M1I90M = 3140296 0 GTCTCTTTTTTTTCATTTATGCATTCATATTCAACAGGTGAGCTTCTGGAGGCTTCTTTGAGTTTTGTCATGGTGGTTTGAAATGTTCCAATGAGA FFFF<FFFFFFFFAFFAFFFFFFAFFFFFFFFAFFFFA<FFFF<.FFFFFFFF)7FFFFFFF.AFFF<FFFFF.FF))FFFFFFFAFFFFFFFAFF AS:i:-33 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:24A9T1A4T40A12 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22510:19317:1639 153 chr10 3140491 3 23M1D93M = 3140491 0 GCTTCCACATGGGATTCAGGTTGTTTTTTATAACCTCTGTTCGGTGAACCATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGA FA<<FF7<F7FFFF).FFFF7FF<F7AFAFFFAFFAFFFAAFFFAFFFFFFFFFF<<FFAFFFFFFFFFFFFFFFFF<FF<FFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFF AS:i:-42 XN:i:0 XM:i:7 XO:i:1 XG:i:1 NM:i:8 MD:Z:6C1G14^T11T0C6A20T0C51 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:13206:16212:9184 153 chr10 3140541 24 116M = 3140541 0 CATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGT FF<FFFFFFF7F<FFFFFFFFFFAFFFF<<FFFFF<FF<FFFFFFFF<FFFFFFFAFFFFFAFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-25 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:14T0C77T1C4G15 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:13504:14353:9068 153 chr10 3140544 23 116M = 3140544 0 CAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGTTAT FFFFAFF..FFFF.AFFF.F.F.FFAAF.FFFFFFFFFA<7A7)AF)FFFFF<FFFF7A<FFFFAFFFFFAFFFFFFF..F7F<FFFAAFFFA.FFFAAFFAFFFFFF.F<F7AFF AS:i:-30 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:11T0C77T1C4G16G1 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:23503:14378:11418 153 chr10 3140637 0 115M = 3140637 0 TTCAAACAATACCACTCTGTTATCCTTTATTTCTTCTGCTGAAATCGTAATACTCCCTTTCCCTGCAGGTTTGCCATTTTTGAGCACCAGTGGTCGAGTTAGCTTCTTACTGGAA AFFFA7FF<<FFFFFFF7FFFFAFFAF.FFFFFAFF7FFFFFFFFAFAF<FFFAF.FFAFFFFFFFFFFF.FFFFFFF<7FFFFFAF7FFFFFAAFFFFFFFAFFFFFFFFFFFA AS:i:-65 XN:i:0 XM:i:13XO:i:0 XG:i:0 NM:i:13 MD:Z:4G16G15A7T1C3G6C6A26A1T0A5T10T2 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:23310:16699:2534 153 chr10 3361500 42 115M = 3361500 0 TTTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA FFAAAF7F7<FFA.FFF<FF.F<.7FFFFFFFF.FFFFAFFFFAAFFFFFFFFFFFFFFFAFFAFFFFFFAFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:11207:15928:8706 153 chr10 3361501 42 114M = 3361501 0 TTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA AAFAFAFFFFFFAFFAFFFAAFFF<F.FFFFF.FFFFFFFFFF<FFFFFFFFFFAFFFFFFFFFFFFFFFFFF7FFFFFF<FFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:12108:17217:19287 137 chr10 3367730 42 89M = 3367730 0 TACACACATACACATGATGGAGGGAGAAAAATACCCAAAGCCAAATGGTTTCCCGAGAGTCTTAAGAAACAAGGGTTCTCAATGAAGTG <AF.F.FFF<FFA7FFAAFFFFFFFF<<.A.F.))F...FFF)).FFFA<A.AFF.F<<AF.A.7<...A..FAF)7AFA<7.F.<A7< AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:33A55 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:12601:5782:3269 137 chr10 3381708 42 111M = 3381708 0 TTTATTTTGTATAATGACGTGATCCTAAAGTATCATTAAGTAGAATCACTCAACCAAGTGCCACAAAACTTTGGGATATAGCTAGTTACCCCATTTTTAAAACGAGAAAGC FFA)F7<.)AFA.FFA<F.)AFAFF.7A7FFFFF<FF.FFA7FFFFFFAFFF<FF.<FFFFF.F.A.)FF<FA)FF<7AA.AF.FFAF<)AFA7FFAA7.F)F<FA<FFFF AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:19G91 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22407:23046:19729 137 chr10 3382964 42 116M = 3382964 0 GCACAAGATGTTATCTATGATGGACAGAAGTAAAGTAACTCTTTTACTAAATAAAATTGGAAAACCCATTAACTTGCCTGCTTATATTAGCAATTACTTGTCTTCATGTCTTCAAC FFFFFFFFFFFFFFFF.FFFFFFFF<FF<FAFFFFFFFFAFFFFFAFF.FFFAFFAFAFFAFF)FFFFFF.AFFFFAFFAAFFFFAFFFFF.<FFFFFFAAFFFAF<FAFFF<..F AS:i:-3 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:16T99 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:22412:2081:16239 137 chr10 3383028 42 116M = 3383028 0 GGCATTAACTTGCCTGCTTATATTAGCAATTACTTGTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACATAATTAGATGTAATTGG FFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFA<FFAFFFFF<)FFFFFF<FFFFF7FFFFFFFAFAFFAAFFF.7FFFF.AFFFF AS:i:-10 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:0C0C114 YT:Z:UP XF:Z:__no_feature

NS500688:2:H3HYJBGXX:4:13503:15452:9506 137 chr10 3383031 42 32M1D64M = 3383031 0 ATTAACTTGCCTGCTTATATTAGCAATTACTTTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACAT FFFFAFFFFFFFFFFFFFFFFFFFFFFFFFF7FFFFFFF.FA.FFF.FFFFFFFFFFFFFFA.FFFFAF<FF.FFFFFFFFFFFFFFFFA<F<FFA AS:i:-8 XN:i:0 XM:i:0 XO:i:1 XG:i:1 NM:i:1 MD:Z:32^G64 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:22211:5786:4879 137 chr10 3383034 42 116M = 3383034 0 AACTTGCCTGCTTATATTAGCAATTACTTGTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACATAATTAGATGTAATAGGTATGGT <FFFFFFFFFFFFAFAF.AFFFFAFAFFFFFFFFFFFFFFAFFFFF.FFFFFF<<F<.F.)FFF.FF7.F<FFFFAFAAFFAFA7F7<.FAFFFFFFA.F.F<FAAF)F<F<FF.F AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:107T8 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:13412:4276:11240 137 chr10 3383035 42 116M = 3383035 0 ACTTGCCTGCTTATATTAGCAATTACTTGTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACATAATTAGATGTAATTGGTATGGTC FFFFFFFFFFFFFFFFFFFF<FFFFFFFFAFFFFFAFFFFFFAFFFFFFFFFFFFAAFFFFFFFFF<AFFFFFFFFFFF7FFFF.FF<FFF7FAFFF<FFFFFFFFF.<F7FFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:12610:9167:7416 137 chr10 3383045 42 116M = 3383045 0 TTATATTAGCAATTACTTGTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACATAATTAGATGTAATTGGTATGGTCATTTCATTCC FFFF<FFFFFFFFFFFF<<FF<FFFFAFFA<FF<FFFA<FAFAFFAFA<FFFFAFA7A)FFFFFAFF<FFFFAFFAAA)FF7F.AFA.F.FAF.AFA7FA<FA.FA.F<AAAAFF7 AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:22606:26008:10720 137 chr10 3383176 42 51M = 3383176 0 AATTACATCTATTTAAAGCATAGAAGTAAGGTGCAGGGTTTTCAGACTGGT FFFFFFFFFFFFFFAFFFFAFAF<FFFAFFFFFFAFAFFFFFFAF<FFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:51 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:11506:12922:5257 137 chr10 3383177 42 50M = 3383177 0 ATTACATCTATTTAAAGCATAGAAGTAAGGTGCAGGGTTTTCAGACTGGT FFFFFAFFA<FAF.AFFFAF.F<AFFAAFAF.F<F.F<F.AF.F.FF.AF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:50 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:21511:14617:12634 137 chr10 3387001 42 116M = 3387001 0 GTTATTTTTGGAAATTTTATTTCTGGGATAGCTTTGTTTATCATACTTATTTAAATTTTTTTCCTATGTGTGACAGTCTGGGCAAACATGCAGTTAGCCTAGGAATTAGAGCTTTT FFFAFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFFF<FFFFFFFFFFFF<FFFFFFAFFFFFFAFFAAFFFFFFFFF<F.F<FF.FFAF<F<F7FFFFAA AS:i:-5 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:1G114 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:23506:12775:14852 137 chr10 3387019 40 116M = 3387019 0 ATTTCTGGGATAGCTTTGATTAAAATACTTATTTAAATTTTTTTCATATGTGTGACAGTCTGGGCAAAAATGCAGTTAGCCTAGGAATTAGAGCTTTTATATTGCTATCAGAACAT )7FF..)))AF7.FF)7F)FF7)).7<AFF.F.<AFF7F<AFFFF.FF..F.FFFAF.).F)..FFFF)FA.FF.<FFA7<)<))<.F<<FA.<FF7)F))A...FA.)A7F.AFF AS:i:-14 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:18T3T0C21C22C43C3 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:13302:7128:6929 137 chr10 3387020 42 116M = 3387020 0 TTTCTGGGATAGCTTTGTTTATCATACTTATTTAAATTTTTTTCCTATGTGTGACAGTCTGGGCAAACATGCAGTTAGCCTAGGAATTAGAGCTTTTATATTGCTATCAGACCATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFF<FAFFFFFFFFFAFFFF<FF<FFAFFFFAAAFFFFF<F<FAAFF<FFFFF<.FFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:11308:7097:4068 137 chr10 3388008 42 115M = 3388008 0 ATCAACCATTTGGGGCTATTGGAGAACCTCAAAGAAAGAATAAGTGACTTTGGGGTCTCAGAGAGATCCTGGTGCTGTGTCTGTTCTTTGCTGGAGAGAGTCTTGGTGGTTAGAC FFFF.FFFFFFFFFFFF<FFFFFFAAFFFFFFFFFFFFFFF.FFFFFFFFFFFFFFFAFFFFF<A7AFAFAFFFFFFF.FFF<FAFFFFFFFFAF<FF)<FFFFAFFFFFF.FAA AS:i:-3 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:41T73 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:12410:3290:5873 137 chr10 3399303 42 28M = 3399303 0 GAGATTTTAAAAAGGAGATGAGTTGATG FFFFFFFF.AFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:28 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:13409:20061:19478 137 chr10 3401968 42 116M = 3401968 0 AAAAAAGCAAAGCAGAATACTATATTCCAACATGGCTTTACACTCTAGGCAGAGGTCAGTTTATTATCAAGGTTTCTTATTTTGTTAGGCAAATTGTTATAATTGGATGTTCTGTC AAFAFAFFFFFFFFF7<FFFFFFFAFF.).FFFFFF..FFFFFFFF..FFFFFAFFF)A)F)<AFFFFAFAF7FFFF.FF77.FF77.FAAFFFAF7FFFFA)FFA7<.FFFA.FF AS:i:-9 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:46T10T48T9 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22609:22407:4141 137 chr10 3401968 42 116M = 3401968 0 AAAAAAGCAAAGCAGAATACTATATTCCAACATGGCTTTACACTCTTGGCTGAGGTCTGTATATTATCAAGGTTTCTTATTTTGTTAGGCAAATTGTTATAATTGGTTGTTCTGTC FFFFFFFFFFFFFFFAFFFFFFFFFFFF<FFFFF<FFFFFFFFFFFFFAF)FFFFAFFFF)FFFF.FF.FFAF.FFFFF<FFFFFFFFFFFFFFFAAA.F77FFAFFFF7<AF<FF AS:i:-4 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:50A9T55 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:23209:19543:12018 137 chr10 3401968 42 116M = 3401968 0 AAAAAAGCAAAGCAGAATACTATATTCCAACATGGCTTTACACTCTTGGCAGAGGTCTGTTTATTATCAAGGTTTCTTATTTTGTTAGGCAAATTGTTATAATTGGTTGTTCTGTC FFFFFFFFFFFFFFFFAFFFFFF<FFFAA)FFFFFFFFF<FFFFFFAF<F<FFFFFFFFA.FFFFFFFFFFFA.FFFFFFFFFFF).<AF7FFFAFFFAFFFFFFFFF<FFAFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:13603:1940:7750 137 chr10 3451792 42 115M = 3451792 0 GCCGCACACTGGCAAGTATTTAGCCACAGCAATCATTCTTCAAGATCCAACATCACTTCCTTCAGGAAAGTGGAGATCCCATGTTCCACTCGCTATCACCCATCGTATAAAAAAA FFFAFFFFFFFFFFFFFF7FFFF.AFFFFF.AAFFFFFFFF<FFFF7F.FFFFF)FFFFFF<F<<FFA<FFFF<F.AFFFFFF.FFFFFFAAFA.AFAFFAFFFFFFF.FFFFFF AS:i:-8 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:23A89T1 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:11210:16682:1643 137 chr10 3463189 42 116M = 3463189 0 GAGGGAAGAGCAAGAAAGCTTGGGCTGGGAAGTTTAAGCATTTCCTGTTTTCCTAAAATCCCTTTCTCATTTTACATTGGAGTTTAACACATACAAGAGCATTCATTAACTTCAGC F<A<FAAFF7FAAFFF.AF7AA).FFF.FAAF<FFF<A<FF)FFFFF))F<FFFF<FF<F<FFF<FAA7F)7F<F.F<7F<FFFA<FFAFAFAFA<F.FFFAFF.F<.FFFF7FA< AS:i:-3 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:97G18 YT:Z:UP XF:Z:__no_feature

NS500688:2:H3HYJBGXX:1:13112:5151:3196 137 chr10 3463382 2 96M = 3463382 0 AACAGAGGAATTCATCAGAGAGCTGCTCTCCCGGATAAGAGGCATGAGGAAGCTGAGCCCTCCGCAGAAGAAGAGCGTATGATTTCACTACAGGGT FFFFFFFFFFFFFFFFFFFF<AFFFFFFFFFFAFFF.FFAAFFFFFFFFFFFFFFFF7FFAF.<F.AF <F7FF))FFF<FFF<FFF7FF.F7FAFA AS:i:-25 XS:i:-27 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:0T0T0T0T0T91 YT:Z:UP XF:Z:Ppp1r14c

NS500688:2:H3HYJBGXX:2:12309:8626:11600 137 chr10 3463384 11 95M = 3463384 0 CAGAGGAATTCATCAGAGAGCTGCTCTCACGGATAAGAGGCATGAGGAAGCTGCGCCCTCCGCAGAAGAAGAGCGTATGATTTCACTACAGGGTA A<F<AAFAFFF7FF)F)F.AFFFA.F7F)FFFF.AFFA<FAFA<7FFF<7F7F)FFFF<FFAFAF7FFF.F)<FAFAFA.FFF7AFF<F.F7<FF AS:i:-18 XS:i:-31 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:0T0T0T25C24A41 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:1:22101:24997:1638 137 chr10 3463400 35 114M = 3463400 0 AGAGCTGCTCTCCCGGATAAGAGGCATGAGGAAGCTGAGCCCTCCGCAGAAGAAGAGCGTATGATTTCACTACAGGGTAAAATTCTCTCGGCGATGAAGAAAGCAACCTGGGAT FF7FFFFFFFFFF.FFA<.FF.FFFF.FFFFFFFFFF7FF<FFFFAF7F<FFFAFAFFFFA.FA.AF..F<FF.FFFF..<.<FF7FFFFFFFF7FAFF).7FF..AFFFFFFA AS:i:0 XS:i:-39 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:1:23106:16253:7204 137 chr10 3463804 37 116M = 3463804 0 ACAGGGAAGATGGTTTTCTTATGGTCAGTTTGGTAAGACGTTTTAAGTTGTAAATTTGACTTCAATGTCAAAGCTATCTCATGCTTGTATATATCTACAGACACTAAGTTAAAACT FFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFFAFFFFFFFAFFFFFFFFFFFAF<FFFFAFFFFF.FFFFFFFFFFFFFFFFAFFAFAAFFFFF<<FFFFF<FAFFFFF.FF AS:i:0 XS:i:-52 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:3:13506:1834:16125 137 chr10 3463952 17 113M = 3463952 0 TAAGCCTGTGCTTCCCTATTAGATGTGGTCACAACACACGTGCTATCAAAAGGTCTCCAGACATTCTTCAGACAAATGTCGTCCACCTTCAGCTTCCACTTTCTGCAACAAAG FF<.FFFFFFFFFFFFF7F..FFF.AFFFFFFFFF.FAFFFAFF.FF.F7FFF)F<FFFFFFAFFFAFFF.FFFFFF7FFFFF<.FFFF<)<F)<FF)AFFFFF.F..FFFFF AS:i:-5 XS:i:-21 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:44T48A19 YT:Z:UP XF:Z:Ppp1r14c

NS500688:2:H3HYJBGXX:2:13110:13087:7560 137 chr10 3463977 30 95M = 3463977 0 TGGTCACAACACACGTGCTTTCAAAAGGTCTCCAGACATTCTTCAGACAAATGTCGTCCACCTTCAGCATCCACTTTCTGCAACAAAGTGAAAGA FFFFFAF<.F)F<FFFFFF7FFFFFFFFFFFFFFFFFFFAFFFFFAFFFF7FFFFFFFF.FFFFFFFF.FFF7FF<FFFFFFFFAFFAF<FFFFF AS:i:0 XS:i:-10 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:95 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:2:23212:12806:5643 137 chr10 3464184 34 116M = 3464184 0 TACTTATTGCCTCATTGTGGTTTTTTAAAGTTGTATGAACTCTTAATTTTGAAAACCAGCCTGATTGATAAAGAAATACCTACACTTTTTGAACACAGTTTTGCAATCAGAAAAGG FFFFFFFFFFFFFFFFFFFFFFFFFF.FFFFFFFFFFFFFFFFFAFFFFFFFFAFFFFFFFFFFFFA.<<FF<FAFAFFFFFFFFF<FFAA.FFFFAFF7FF<F.AFA.<F.F<F< AS:i:0 XS:i:-34 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:2:12102:20330:1921 137 chr10 3464514 31 116M = 3464514 0 AGTCTGGTTTCATCTCTGGTTTCTCTCAGTGGTTCATGGTCCTGATTGTGACCTTGATTTCCATGTTAGGAGCTAGGTCTGATGACCTACCCTAACCCTACAGCTACATACCAGCT FFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFAAFFAFFFFFFAFFFFFFAFFFFFFF<FFFFF<FFAFFFFFFAFFA AS:i:0 XS:i:-18 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:3:23604:10184:2106 137 chr10 3464515 31 116M = 3464515 0 GTCTGGTTTCATCTCTGGTTTCTCTCAGTGGTTCATGGTCCTGATTGTGACCTTGATTTCCATGTTAGGAGCTAGGTCTGATGACCTACCCTAACCCTACAGCTACATACCAGCTC FFFFFFFFFFAFFFAFFFF<FAFFFAF<F<FFFFFFAFFFFF<<FFAFFAFFAF<F7FFFF7F.FFFFF.AFF<FFFFFF)FA)FFF.FFFF7)FFFFAAAFFFFFFFFFFA<FFF AS:i:0 XS:i:-17 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:4:11506:13259:19980 137 chr10 3464571 6 112M = 3464571 0 TTTCCATGTTAGGAGCTAGGTCTGATGACCTCCCCTAACCCTACAGCTACATACCAGCTCTATGAGTAGAATTTAACTTCATATGGGATGTAGGCAGGGTAATAGTTGCGTA FAFFF)FFFFAFF7FFFFFAFFFFAAF7FAF)FF<F7)77FFFFFFFAFFFFFF7)AFFFA.FF)F7A<F<FF)77)FFF<FFF.<FFFFFAFFF.AFFFFA.F<FAA)F77 AS:i:-14 XS:i:-20 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:31A29T11A2A25A5A3 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:2:12307:22275:8351 137 chr10 3464726 12 115M = 3464726 0 TTCCAGGAAGCCCCTGCTGCCTCCCCCTGGAAACCCTGCCTGCCGTGAGAAGTCTCCAGGTGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGAGCTTTGAACAT 7FFFAAFFFFF7)FFFFFFF7FAF.<7F7.<FFF<FF7<FFAA.)F.FA<FAFFFFFFF<FFFFFFFFFFFFFF.FAFA<FFF<FF.A7<FF<.FFFAA)F.F).AFF7AF.)FF AS:i:-6 XS:i:-16 XN:i:4 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:0N0N0N0N99T11 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:1:12208:21960:17118 137 chr10 3464736 6 116M = 3464736 0 CCCCTGCTGCCTCCCCCTGGAAACCCTGCCTGCCGTGAGAAGTCTCCAGGTGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGTGCTTTGAACATATTTTATTTTT FF<FAFFFFFFFFFFFFFFFFF.FFFFFFFFFFFFAFFFFFFFFFFFAFFFFFFFFFFFFFF...FFFFFF.FFFFFFFFAFF<<7FFAAAFFFFFFFF<F.F<FFFFFFFAFFFF AS:i:0 XS:i:-5 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:2:H3HYJBGXX:1:13312:17008:11028 137 chr10 3464737 6 96M = 3464737 0 CCCTGCTGCCTCCCCCTGGAAACCCTGCCTGCCGTGAGAAGTCTCCAGGTGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGTGCT FFFFFFFFFFFFFFFFFFFFF.FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFAFFFF AS:i:0 XS:i:-5 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:96YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:4:23501:23438:14742 137 chr10 3464759 6 116M = 3464759 0 CCCTGCCTGCCGTGAGAAGTCTCCAGGTGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGTGCTTTGAACATATTTTATTTTTTAAAAGAACTATGTTTGTGAATT F)FFFFF<FAFFFFFFF.FFFFFFFFFFFFFFFFFFF7AAFFFFFAF.<FFFFFFFF)FFF)AFFFF<..)FFFAFFAAFFFFF<FFF.FFFFFAF7A.<FFAFFA7.<F.FAFF< AS:i:0 XS:i:-5 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:4:22608:22580:1379 137 chr10 3464786 1 115M = 3464786 0 TGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGTGCTTTGAACATATTTTATTTTTTAAAAGAACTATGTTTGTGAATTTTCCGTATACTGGCAAGCTTTTGAAA FFAFFFFAFFFFFFFFFFFFFFFFFF.FFFFFFAFAFA7F.7AFFFFFFFFFFAF.FFFFAFFFFFF.A.<AFFFFAAFFF<F)7FFFF<AF.7F.FAFAF7<).FFFFAA<<.< AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:1:21110:15851:11639 137 chr10 3464825 1 116M = 3464825 0 AGGGTGCTTTGAACATATTTTATTTTTTAAAAGAACTATGTTTGTGAATTTTCCGTATACTGGCAAGCTTTTGAAAATGTATTTAATTTTGTAATGTTTACCAATGATTTATTTAC FFFFFFFFFFFAAFFFFFFAF.FFFFFFFFFFFAFFFAFFFFF.F<AFFFFAFFFFAFFFFFFF7FAFFF<FFAFFFFFFFFFA7FFFF<.A<FFFFFFFFF.<FF.FFFAF<AAA AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:2:H3HYJBGXX:4:13605:2329:17680 137 chr10 3464850 1 96M = 3464850 0 TTTAAAAGAACTATGTTTGTGAATTTTCCGTATACTGGCAAGCTTTTGAAAATGTATTTAATTTTGTAATGTTTACCAATGATTTATTTACAAGCT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFAFFFFF<FFFFFAFFFFFFFFFFF.FFFFFFFFFFFFF.F.FA AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:96 YT:Z:UPXF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:3:21611:10784:7699 137 chr10 3464862 1 114M = 3464862 0 ATGTTTGTGAATTTTCCGTATACTGGCAAGCTTTTGAAAATGTATTTAATTTTGTAATGTTTACCAATGATTTATTTACAAGCTATTTACTCAAATAAAGGGAGCTGCTTACAA FFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFF<AFFFFFFFFFFFFFAAFFFFFFFFFFF.FFFFFFFFFFFFF.FFFFFFFFFFFF.FFF<FAFF<FAFAFFFAFFAF AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:2:23204:9837:15537 137 chr10 3464915 1 61M = 3464915 0 GTAATGTTTACCAATGATTTATTTACAAGCTATTTACTCAAATAAAGGGAGCTGCTTAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFAAFFAFFFFFFFFFFFFFAAFF AS:i:-10 XS:i:-10 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:58C1A0 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:3:22403:6146:13471 137 chr10 3555451 42 116M = 3555451 0 AGTTTTCTCTGAGTTTGCTTTCCTTCCCTCCACCTGTGATAAGTATGGTCCCTGGGGCTTTACTGAAACGGGGTTTCTTTTAATGCTATTGTGGCTTTGGTAGGGAATGGGAACCT FFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFAFFFFFFFFFFFFFFFFFF7FFFF.FFFFF<FFFFFFFFFFF7FFFFFFF.<FFFFAFFFFF<FFFFFFFFFFFFFFFFFF<FFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:2:H3HYJBGXX:1:12302:2073:2501 137 chr10 3724572 42 96M = 3724572 0 GCAGAGCATGCTCCAGCTGCTGTGCTGAGCCACTGAATTAATGGTACTATGTGGTGCAGTGATAGACAAACAACCACAGCAAGCAAGTGGACTCTC FFFF7FFFFFFFFFFAFFFFFFFFFFFAFFFFFFFFFFF7FFFFF7FFFFFFFFFFFFFFFFFFF7FFA)F.)FFFFFFFFF<FF7FFFF<FFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:96 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:12402:13489:9837 137 chr10 3741923 42 115M = 3741923 0 TTCCTGACTTAGCTTTTTGTCTCTTGGAATTGCACCCCGTCTAAGCCGTTAAAACCGACCAGGAAGAAACTTTCTTTTCCAGAGTTCACTGACCTCCCTGGCTTCATCCGCAAGT FFFFFAFFAFFFFFFFFA<FFFFFFA.FFFFFF7F7FFFFFFFAF7FFF.FFFF)FFAFAAF.<).FFFFFFFFF)F7.FFF.F)7F.FFFF.FFF7FFA.FFFF...<.AF<FF AS:i:-3 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:105C9 YT:Z:UP XF:Z:__no_feature

On Mon, Apr 27, 2015 at 11:30 PM, Li Shen notifications@github.com wrote:

I'm not sure what is going on. Can you show us your bam's head?

Li

On Mon, Apr 27, 2015 at 10:43 AM, orzechoj notifications@github.com wrote:

Hi Shen Lab,

I have a similar problem: Running ngs.plot.r produces plots where all values are 0. I run the following command

ngs.plot.r -G mm10 -R genebody -C my.bam -O outPrefix

(I installed the mm10 genome, and I didn't get any problems there.) When I run the program on the supplied example

ngs.plot.r -G hg19 -R tss -C hesc.H3k4me3.1M.bam -O hesc.H3k4me3.tss -T H3K4me3 -L 3000 -FL 300

everything worked ok.

I checked that the chromosome names in the mm10 genome.coord object are the same as in the bam file, e.g. "chr12", so for me that's not the problem. I also tried "-R tss", and/or "-D refseq" and got similar results. Also, "-MQ 1", "-F rnaseq" and "-SS same" didn't change anything.

(I ran this using ngsplot-2.47 as well as the ngsplot-develop version downloaded from gitHub 4/26-2015.)

Do you have any ideas what I could try next?

kind regards /Jakub

— Reply to this email directly or view it on GitHub < https://github.com/shenlab-sinai/ngsplot/issues/39#issuecomment-96681168> .

Li Shen, Ph.D. Computer Science Assistant Professor Department of Neuroscience Icahn School of Medicine at Mount Sinai New York, NY 10029 ################# ngs.plot - the coolest NGS plotting tool: https://code.google.com/p/ngsplot/ Want ChIP-seq differential analysis? Use: https://code.google.com/p/diffreps/

— Reply to this email directly or view it on GitHub https://github.com/shenlab-sinai/ngsplot/issues/39#issuecomment-96827588 .

lishen commented 9 years ago

I think I found the reason. The SAM flags in your bam file are either 137 or 153, which means "read paired", "mate unmapped", "second in pair" and "read reverse strand" (optional). Because ngs.plot understands paired-end alignments and sees these reads as inappropriate, they are all filtered.

What you need to do is to modify the SAM flags to fool the program into believing that they are single-end alignments. An option is to use bedtools to convert your bam into bed and then convert it back into bam. During this conversion, the unnecessary flags will be discarded.

Li

On Tue, Apr 28, 2015 at 3:03 AM, orzechoj notifications@github.com wrote:

Hi Li, thank you for the quick reply,

The bam file I'm using is a bit weird. The library was generated using a special protocol, so what I have is paired end data, but only the second read in each pair is used. The data were mapped with bowtie2. Below are the first 10 lines, apologies for the lousy formatting.

best, Jakub

$ samtools view R1_formated_mapped_filtered_annotated_SMALL_SORT.bam | head -n 10

NS500688:7:H5FYJBGXX:2:13110:2209:5781 153 chr10 3139861 0 76M1I37M = 3139861 0

ATATTTGGAGATAAATTAGTTGGTCCATAGAGTCTTATCTGGGGAAGACAGGTCCGATAAGCCTCTACAATGCCTTGGATTCCATTGCAATAGGGGTTCGATGGGTTAAAGCTG

7FA)FFFFAAF)F.F7))<)<.7)FF..AFF<)FF<FAA7FFFFFFAF.FFFFA<FF7F<FFA<F<FAFFFFFFFF<FFFFFFFFFFFFFFFFFFF.AFFFFFFF7FFFFFFFF AS:i:-53 XN:i:0 XM:i:11 XO:i:1 XG:i:1 NM:i:12

MD:Z:3A7A10A4C13A12T12T32G9T0C0C0 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:22103:17410:17927 153 chr10 3140296 8 5M1I90M = 3140296 0

GTCTCTTTTTTTTCATTTATGCATTCATATTCAACAGGTGAGCTTCTGGAGGCTTCTTTGAGTTTTGTCATGGTGGTTTGAAATGTTCCAATGAGA

FFFF<FFFFFFFFAFFAFFFFFFAFFFFFFFFAFFFFA<FFFF<.FFFFFFFF)7FFFFFFF.AFFF<FFFFF.FF))FFFFFFFAFFFFFFFAFF AS:i:-33 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:24A9T1A4T40A12 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22510:19317:1639 153 chr10 3140491 3 23M1D93M = 3140491 0

GCTTCCACATGGGATTCAGGTTGTTTTTTATAACCTCTGTTCGGTGAACCATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGA

FA<<FF7<F7FFFF).FFFF7FF<F7AFAFFFAFFAFFFAAFFFAFFFFFFFFFF<<FFAFFFFFFFFFFFFFFFFF<FF<FFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFF AS:i:-42 XN:i:0 XM:i:7 XO:i:1 XG:i:1 NM:i:8 MD:Z:6C1G14^T11T0C6A20T0C51 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:13206:16212:9184 153 chr10 3140541 24 116M = 3140541 0

CATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGT

FF<FFFFFFF7F<FFFFFFFFFFAFFFF<<FFFFF<FF<FFFFFFFF<FFFFFFFAFFFFFAFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-25 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:14T0C77T1C4G15 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:13504:14353:9068 153 chr10 3140544 23 116M = 3140544 0

CAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGTTAT

FFFFAFF..FFFF.AFFF.F.F.FFAAF.FFFFFFFFFA<7A7)AF)FFFFF<FFFF7A<FFFFAFFFFFAFFFFFFF..F7F<FFFAAFFFA.FFFAAFFAFFFFFF.F<F7AFF AS:i:-30 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:11T0C77T1C4G16G1 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:23503:14378:11418 153 chr10 3140637 0 115M = 3140637 0

TTCAAACAATACCACTCTGTTATCCTTTATTTCTTCTGCTGAAATCGTAATACTCCCTTTCCCTGCAGGTTTGCCATTTTTGAGCACCAGTGGTCGAGTTAGCTTCTTACTGGAA

AFFFA7FF<<FFFFFFF7FFFFAFFAF.FFFFFAFF7FFFFFFFFAFAF<FFFAF.FFAFFFFFFFFFFF.FFFFFFF<7FFFFFAF7FFFFFAAFFFFFFFAFFFFFFFFFFFA AS:i:-65 XN:i:0 XM:i:13 XO:i:0 XG:i:0 NM:i:13 MD:Z:4G16G15A7T1C3G6C6A26A1T0A5T10T2 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:23310:16699:2534 153 chr10 3361500 42 115M = 3361500 0

TTTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA

FFAAAF7F7<FFA.FFF<FF.F<.7FFFFFFFF.FFFFAFFFFAAFFFFFFFFFFFFFFFAFFAFFFFFFAFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:11207:15928:8706 153 chr10 3361501 42 114M = 3361501 0

TTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA

AAFAFAFFFFFFAFFAFFFAAFFF<F.FFFFF.FFFFFFFFFF<FFFFFFFFFFAFFFFFFFFFFFFFFFFFF7FFFFFF<FFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:12108:17217:19287 137 chr10 3367730 42 89M = 3367730 0

TACACACATACACATGATGGAGGGAGAAAAATACCCAAAGCCAAATGGTTTCCCGAGAGTCTTAAGAAACAAGGGTTCTCAATGAAGTG

<AF.F.FFF<FFA7FFAAFFFFFFFF<<.A.F.))F...FFF)).FFFA<A.AFF.F<<AF.A.7<...A..FAF)7AFA<7.F.<A7< AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:33A55 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:12601:5782:3269 137 chr10 3381708 42 111M = 3381708 0

TTTATTTTGTATAATGACGTGATCCTAAAGTATCATTAAGTAGAATCACTCAACCAAGTGCCACAAAACTTTGGGATATAGCTAGTTACCCCATTTTTAAAACGAGAAAGC

FFA)F7<.)AFA.FFA<F.)AFAFF.7A7FFFFF<FF.FFA7FFFFFFAFFF<FF.<FFFFF.F.A.)FF<FA)FF<7AA.AF.FFAF<)AFA7FFAA7.F)F<FA<FFFF AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:19G91 YT:Z:UP XF:Z:__no_feature

On Tue, Apr 28, 2015 at 9:00 AM, Jakub Westholm jakub.westholm@gmail.com wrote:

Hi Li, thank you for the quick reply,

The bam file I'm using is a bit weird. The library was generated using a special protocol, so what I have is paired end data, but only the second read in the pair is used. The data were mapped with bowtie2. Below are the first 50 lines, apologies for the lousy formatting.

best, Jakub

$ samtools view my.bam | head -n 50

NS500688:7:H5FYJBGXX:2:13110:2209:5781 153 chr10 3139861 0 76M1I37M = 3139861 0

ATATTTGGAGATAAATTAGTTGGTCCATAGAGTCTTATCTGGGGAAGACAGGTCCGATAAGCCTCTACAATGCCTTGGATTCCATTGCAATAGGGGTTCGATGGGTTAAAGCTG

7FA)FFFFAAF)F.F7))<)<.7)FF..AFF<)FF<FAA7FFFFFFAF.FFFFA<FF7F<FFA<F<FAFFFFFFFF<FFFFFFFFFFFFFFFFFFF.AFFFFFFF7FFFFFFFF AS:i:-53 XN:i:0 XM:i:11XO:i:1 XG:i:1 NM:i:12 MD:Z:3A7A10A4C13A12T12T32G9T0C0C0 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:22103:17410:17927 153 chr10 3140296 8 5M1I90M = 3140296 0

GTCTCTTTTTTTTCATTTATGCATTCATATTCAACAGGTGAGCTTCTGGAGGCTTCTTTGAGTTTTGTCATGGTGGTTTGAAATGTTCCAATGAGA

FFFF<FFFFFFFFAFFAFFFFFFAFFFFFFFFAFFFFA<FFFF<.FFFFFFFF)7FFFFFFF.AFFF<FFFFF.FF))FFFFFFFAFFFFFFFAFF AS:i:-33 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:24A9T1A4T40A12 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22510:19317:1639 153 chr10 3140491 3 23M1D93M = 3140491 0

GCTTCCACATGGGATTCAGGTTGTTTTTTATAACCTCTGTTCGGTGAACCATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGA

FA<<FF7<F7FFFF).FFFF7FF<F7AFAFFFAFFAFFFAAFFFAFFFFFFFFFF<<FFAFFFFFFFFFFFFFFFFF<FF<FFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFF AS:i:-42 XN:i:0 XM:i:7 XO:i:1 XG:i:1 NM:i:8 MD:Z:6C1G14^T11T0C6A20T0C51 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:13206:16212:9184 153 chr10 3140541 24 116M = 3140541 0

CATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGT

FF<FFFFFFF7F<FFFFFFFFFFAFFFF<<FFFFF<FF<FFFFFFFF<FFFFFFFAFFFFFAFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-25 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:14T0C77T1C4G15 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:13504:14353:9068 153 chr10 3140544 23 116M = 3140544 0

CAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGTTAT

FFFFAFF..FFFF.AFFF.F.F.FFAAF.FFFFFFFFFA<7A7)AF)FFFFF<FFFF7A<FFFFAFFFFFAFFFFFFF..F7F<FFFAAFFFA.FFFAAFFAFFFFFF.F<F7AFF AS:i:-30 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:11T0C77T1C4G16G1 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:23503:14378:11418 153 chr10 3140637 0 115M = 3140637 0

TTCAAACAATACCACTCTGTTATCCTTTATTTCTTCTGCTGAAATCGTAATACTCCCTTTCCCTGCAGGTTTGCCATTTTTGAGCACCAGTGGTCGAGTTAGCTTCTTACTGGAA

AFFFA7FF<<FFFFFFF7FFFFAFFAF.FFFFFAFF7FFFFFFFFAFAF<FFFAF.FFAFFFFFFFFFFF.FFFFFFF<7FFFFFAF7FFFFFAAFFFFFFFAFFFFFFFFFFFA AS:i:-65 XN:i:0 XM:i:13XO:i:0 XG:i:0 NM:i:13 MD:Z:4G16G15A7T1C3G6C6A26A1T0A5T10T2 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:23310:16699:2534 153 chr10 3361500 42 115M = 3361500 0

TTTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA

FFAAAF7F7<FFA.FFF<FF.F<.7FFFFFFFF.FFFFAFFFFAAFFFFFFFFFFFFFFFAFFAFFFFFFAFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:11207:15928:8706 153 chr10 3361501 42 114M = 3361501 0

TTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA

AAFAFAFFFFFFAFFAFFFAAFFF<F.FFFFF.FFFFFFFFFF<FFFFFFFFFFAFFFFFFFFFFFFFFFFFF7FFFFFF<FFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:12108:17217:19287 137 chr10 3367730 42 89M = 3367730 0

TACACACATACACATGATGGAGGGAGAAAAATACCCAAAGCCAAATGGTTTCCCGAGAGTCTTAAGAAACAAGGGTTCTCAATGAAGTG

<AF.F.FFF<FFA7FFAAFFFFFFFF<<.A.F.))F...FFF)).FFFA<A.AFF.F<<AF.A.7<...A..FAF)7AFA<7.F.<A7< AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:33A55 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:12601:5782:3269 137 chr10 3381708 42 111M = 3381708 0

TTTATTTTGTATAATGACGTGATCCTAAAGTATCATTAAGTAGAATCACTCAACCAAGTGCCACAAAACTTTGGGATATAGCTAGTTACCCCATTTTTAAAACGAGAAAGC

FFA)F7<.)AFA.FFA<F.)AFAFF.7A7FFFFF<FF.FFA7FFFFFFAFFF<FF.<FFFFF.F.A.)FF<FA)FF<7AA.AF.FFAF<)AFA7FFAA7.F)F<FA<FFFF AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:19G91 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22407:23046:19729 137 chr10 3382964 42 116M = 3382964 0

GCACAAGATGTTATCTATGATGGACAGAAGTAAAGTAACTCTTTTACTAAATAAAATTGGAAAACCCATTAACTTGCCTGCTTATATTAGCAATTACTTGTCTTCATGTCTTCAAC

FFFFFFFFFFFFFFFF.FFFFFFFF<FF<FAFFFFFFFFAFFFFFAFF.FFFAFFAFAFFAFF)FFFFFF.AFFFFAFFAAFFFFAFFFFF.<FFFFFFAAFFFAF<FAFFF<..F AS:i:-3 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:16T99 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:22412:2081:16239 137 chr10 3383028 42 116M = 3383028 0

GGCATTAACTTGCCTGCTTATATTAGCAATTACTTGTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACATAATTAGATGTAATTGG

FFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFA<FFAFFFFF<)FFFFFF<FFFFF7FFFFFFFAFAFFAAFFF.7FFFF.AFFFF AS:i:-10 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:0C0C114 YT:Z:UP XF:Z:__no_feature

NS500688:2:H3HYJBGXX:4:13503:15452:9506 137 chr10 3383031 42 32M1D64M = 3383031 0

ATTAACTTGCCTGCTTATATTAGCAATTACTTTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACAT

FFFFAFFFFFFFFFFFFFFFFFFFFFFFFFF7FFFFFFF.FA.FFF.FFFFFFFFFFFFFFA.FFFFAF<FF.FFFFFFFFFFFFFFFFA<F<FFA AS:i:-8 XN:i:0 XM:i:0 XO:i:1 XG:i:1 NM:i:1 MD:Z:32^G64 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:22211:5786:4879 137 chr10 3383034 42 116M = 3383034 0

AACTTGCCTGCTTATATTAGCAATTACTTGTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACATAATTAGATGTAATAGGTATGGT

<FFFFFFFFFFFFAFAF.AFFFFAFAFFFFFFFFFFFFFFAFFFFF.FFFFFF<<F<.F.)FFF.FF7.F<FFFFAFAAFFAFA7F7<.FAFFFFFFA.F.F<FAAF)F<F<FF.F AS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:107T8 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:13412:4276:11240 137 chr10 3383035 42 116M = 3383035 0

ACTTGCCTGCTTATATTAGCAATTACTTGTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACATAATTAGATGTAATTGGTATGGTC

FFFFFFFFFFFFFFFFFFFF<FFFFFFFFAFFFFFAFFFFFFAFFFFFFFFFFFFAAFFFFFFFFF<AFFFFFFFFFFF7FFFF.FF<FFF7FAFFF<FFFFFFFFF.<F7FFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:12610:9167:7416 137 chr10 3383045 42 116M = 3383045 0

TTATATTAGCAATTACTTGTCTTCATGTCTTCAACATCTGTGGCCAATGATCTGTTAATGTCCTTCTCTTTCATTTTGTACATAATTAGATGTAATTGGTATGGTCATTTCATTCC

FFFF<FFFFFFFFFFFF<<FF<FFFFAFFA<FF<FFFA<FAFAFFAFA<FFFFAFA7A)FFFFFAFF<FFFFAFFAAA)FF7F.AFA.F.FAF.AFA7FA<FA.FA.F<AAAAFF7 AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:22606:26008:10720 137 chr10 3383176 42 51M = 3383176 0 AATTACATCTATTTAAAGCATAGAAGTAAGGTGCAGGGTTTTCAGACTGGT FFFFFFFFFFFFFFAFFFFAFAF<FFFAFFFFFFAFAFFFFFFAF<FFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:51 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:11506:12922:5257 137 chr10 3383177 42 50M = 3383177 0 ATTACATCTATTTAAAGCATAGAAGTAAGGTGCAGGGTTTTCAGACTGGT FFFFFAFFA<FAF.AFFFAF.F<AFFAAFAF.F<F.F<F.AF.F.FF.AF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:50 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:21511:14617:12634 137 chr10 3387001 42 116M = 3387001 0

GTTATTTTTGGAAATTTTATTTCTGGGATAGCTTTGTTTATCATACTTATTTAAATTTTTTTCCTATGTGTGACAGTCTGGGCAAACATGCAGTTAGCCTAGGAATTAGAGCTTTT

FFFAFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFFF<FFFFFFFFFFFF<FFFFFFAFFFFFFAFFAAFFFFFFFFF<F.F<FF.FFAF<F<F7FFFFAA AS:i:-5 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:1G114 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:23506:12775:14852 137 chr10 3387019 40 116M = 3387019 0

ATTTCTGGGATAGCTTTGATTAAAATACTTATTTAAATTTTTTTCATATGTGTGACAGTCTGGGCAAAAATGCAGTTAGCCTAGGAATTAGAGCTTTTATATTGCTATCAGAACAT

)7FF..)))AF7.FF)7F)FF7)).7<AFF.F.<AFF7F<AFFFF.FF..F.FFFAF.).F)..FFFF)FA.FF.<FFA7<)<))<.F<<FA.<FF7)F))A...FA.)A7F.AFF AS:i:-14 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:18T3T0C21C22C43C3 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:13302:7128:6929 137 chr10 3387020 42 116M = 3387020 0

TTTCTGGGATAGCTTTGTTTATCATACTTATTTAAATTTTTTTCCTATGTGTGACAGTCTGGGCAAACATGCAGTTAGCCTAGGAATTAGAGCTTTTATATTGCTATCAGACCATT

FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFF<FAFFFFFFFFFAFFFF<FF<FFAFFFFAAAFFFFF<F<FAAFF<FFFFF<.FFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:11308:7097:4068 137 chr10 3388008 42 115M = 3388008 0

ATCAACCATTTGGGGCTATTGGAGAACCTCAAAGAAAGAATAAGTGACTTTGGGGTCTCAGAGAGATCCTGGTGCTGTGTCTGTTCTTTGCTGGAGAGAGTCTTGGTGGTTAGAC

FFFF.FFFFFFFFFFFF<FFFFFFAAFFFFFFFFFFFFFFF.FFFFFFFFFFFFFFFAFFFFF<A7AFAFAFFFFFFF.FFF<FAFFFFFFFFAF<FF)<FFFFAFFFFFF.FAA AS:i:-3 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:41T73 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:12410:3290:5873 137 chr10 3399303 42 28M = 3399303 0 GAGATTTTAAAAAGGAGATGAGTTGATG FFFFFFFF.AFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:28 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:13409:20061:19478 137 chr10 3401968 42 116M = 3401968 0

AAAAAAGCAAAGCAGAATACTATATTCCAACATGGCTTTACACTCTAGGCAGAGGTCAGTTTATTATCAAGGTTTCTTATTTTGTTAGGCAAATTGTTATAATTGGATGTTCTGTC

AAFAFAFFFFFFFFF7<FFFFFFFAFF.).FFFFFF..FFFFFFFF..FFFFFAFFF)A)F)<AFFFFAFAF7FFFF.FF77.FF77.FAAFFFAF7FFFFA)FFA7<.FFFA.FF AS:i:-9 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:46T10T48T9 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22609:22407:4141 137 chr10 3401968 42 116M = 3401968 0

AAAAAAGCAAAGCAGAATACTATATTCCAACATGGCTTTACACTCTTGGCTGAGGTCTGTATATTATCAAGGTTTCTTATTTTGTTAGGCAAATTGTTATAATTGGTTGTTCTGTC

FFFFFFFFFFFFFFFAFFFFFFFFFFFF<FFFFF<FFFFFFFFFFFFFAF)FFFFAFFFF)FFFF.FF.FFAF.FFFFF<FFFFFFFFFFFFFFFAAA.F77FFAFFFF7<AF<FF AS:i:-4 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:50A9T55 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:23209:19543:12018 137 chr10 3401968 42 116M = 3401968 0

AAAAAAGCAAAGCAGAATACTATATTCCAACATGGCTTTACACTCTTGGCAGAGGTCTGTTTATTATCAAGGTTTCTTATTTTGTTAGGCAAATTGTTATAATTGGTTGTTCTGTC

FFFFFFFFFFFFFFFFAFFFFFF<FFFAA)FFFFFFFFF<FFFFFFAF<F<FFFFFFFFA.FFFFFFFFFFFA.FFFFFFFFFFF).<AF7FFFAFFFAFFFFFFFFF<FFAFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:13603:1940:7750 137 chr10 3451792 42 115M = 3451792 0

GCCGCACACTGGCAAGTATTTAGCCACAGCAATCATTCTTCAAGATCCAACATCACTTCCTTCAGGAAAGTGGAGATCCCATGTTCCACTCGCTATCACCCATCGTATAAAAAAA

FFFAFFFFFFFFFFFFFF7FFFF.AFFFFF.AAFFFFFFFF<FFFF7F.FFFFF)FFFFFF<F<<FFA<FFFF<F.AFFFFFF.FFFFFFAAFA.AFAFFAFFFFFFF.FFFFFF AS:i:-8 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:23A89T1 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:11210:16682:1643 137 chr10 3463189 42 116M = 3463189 0

GAGGGAAGAGCAAGAAAGCTTGGGCTGGGAAGTTTAAGCATTTCCTGTTTTCCTAAAATCCCTTTCTCATTTTACATTGGAGTTTAACACATACAAGAGCATTCATTAACTTCAGC

F<A<FAAFF7FAAFFF.AF7AA).FFF.FAAF<FFF<A<FF)FFFFF))F<FFFF<FF<F<FFF<FAA7F)7F<F.F<7F<FFFA<FFAFAFAFA<F.FFFAFF.F<.FFFF7FA< AS:i:-3 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:97G18 YT:Z:UP XF:Z:__no_feature

NS500688:2:H3HYJBGXX:1:13112:5151:3196 137 chr10 3463382 2 96M = 3463382 0

AACAGAGGAATTCATCAGAGAGCTGCTCTCCCGGATAAGAGGCATGAGGAAGCTGAGCCCTCCGCAGAAGAAGAGCGTATGATTTCACTACAGGGT FFFFFFFFFFFFFFFFFFFF<AFFFFFFFFFFAFFF.FFAAFFFFFFFFFFFFFFFF7FFAF.<F.AF <F7FF))FFF<FFF<FFF7FF.F7FAFA AS:i:-25 XS:i:-27 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:0T0T0T0T0T91 YT:Z:UP XF:Z:Ppp1r14c

NS500688:2:H3HYJBGXX:2:12309:8626:11600 137 chr10 3463384 11 95M = 3463384 0

CAGAGGAATTCATCAGAGAGCTGCTCTCACGGATAAGAGGCATGAGGAAGCTGCGCCCTCCGCAGAAGAAGAGCGTATGATTTCACTACAGGGTA

A<F<AAFAFFF7FF)F)F.AFFFA.F7F)FFFF.AFFA<FAFA<7FFF<7F7F)FFFF<FFAFAF7FFF.F)<FAFAFA.FFF7AFF<F.F7<FF AS:i:-18 XS:i:-31 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:0T0T0T25C24A41 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:1:22101:24997:1638 137 chr10 3463400 35 114M = 3463400 0

AGAGCTGCTCTCCCGGATAAGAGGCATGAGGAAGCTGAGCCCTCCGCAGAAGAAGAGCGTATGATTTCACTACAGGGTAAAATTCTCTCGGCGATGAAGAAAGCAACCTGGGAT

FF7FFFFFFFFFF.FFA<.FF.FFFF.FFFFFFFFFF7FF<FFFFAF7F<FFFAFAFFFFA.FA.AF..F<FF.FFFF..<.<FF7FFFFFFFF7FAFF).7FF..AFFFFFFA AS:i:0 XS:i:-39 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:1:23106:16253:7204 137 chr10 3463804 37 116M = 3463804 0

ACAGGGAAGATGGTTTTCTTATGGTCAGTTTGGTAAGACGTTTTAAGTTGTAAATTTGACTTCAATGTCAAAGCTATCTCATGCTTGTATATATCTACAGACACTAAGTTAAAACT

FFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFFAFFFFFFFAFFFFFFFFFFFAF<FFFFAFFFFF.FFFFFFFFFFFFFFFFAFFAFAAFFFFF<<FFFFF<FAFFFFF.FF AS:i:0 XS:i:-52 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:3:13506:1834:16125 137 chr10 3463952 17 113M = 3463952 0

TAAGCCTGTGCTTCCCTATTAGATGTGGTCACAACACACGTGCTATCAAAAGGTCTCCAGACATTCTTCAGACAAATGTCGTCCACCTTCAGCTTCCACTTTCTGCAACAAAG

FF<.FFFFFFFFFFFFF7F..FFF.AFFFFFFFFF.FAFFFAFF.FF.F7FFF)F<FFFFFFAFFFAFFF.FFFFFF7FFFFF<.FFFF<)<F)<FF)AFFFFF.F..FFFFF AS:i:-5 XS:i:-21 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:44T48A19 YT:Z:UP XF:Z:Ppp1r14c

NS500688:2:H3HYJBGXX:2:13110:13087:7560 137 chr10 3463977 30 95M = 3463977 0

TGGTCACAACACACGTGCTTTCAAAAGGTCTCCAGACATTCTTCAGACAAATGTCGTCCACCTTCAGCATCCACTTTCTGCAACAAAGTGAAAGA

FFFFFAF<.F)F<FFFFFF7FFFFFFFFFFFFFFFFFFFAFFFFFAFFFF7FFFFFFFF.FFFFFFFF.FFF7FF<FFFFFFFFAFFAF<FFFFF AS:i:0 XS:i:-10 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:95 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:2:23212:12806:5643 137 chr10 3464184 34 116M = 3464184 0

TACTTATTGCCTCATTGTGGTTTTTTAAAGTTGTATGAACTCTTAATTTTGAAAACCAGCCTGATTGATAAAGAAATACCTACACTTTTTGAACACAGTTTTGCAATCAGAAAAGG

FFFFFFFFFFFFFFFFFFFFFFFFFF.FFFFFFFFFFFFFFFFFAFFFFFFFFAFFFFFFFFFFFFA.<<FF<FAFAFFFFFFFFF<FFAA.FFFFAFF7FF<F.AFA.<F.F<F< AS:i:0 XS:i:-34 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:2:12102:20330:1921 137 chr10 3464514 31 116M = 3464514 0

AGTCTGGTTTCATCTCTGGTTTCTCTCAGTGGTTCATGGTCCTGATTGTGACCTTGATTTCCATGTTAGGAGCTAGGTCTGATGACCTACCCTAACCCTACAGCTACATACCAGCT

FFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFAAFFAFFFFFFAFFFFFFAFFFFFFF<FFFFF<FFAFFFFFFAFFA AS:i:0 XS:i:-18 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:3:23604:10184:2106 137 chr10 3464515 31 116M = 3464515 0

GTCTGGTTTCATCTCTGGTTTCTCTCAGTGGTTCATGGTCCTGATTGTGACCTTGATTTCCATGTTAGGAGCTAGGTCTGATGACCTACCCTAACCCTACAGCTACATACCAGCTC

FFFFFFFFFFAFFFAFFFF<FAFFFAF<F<FFFFFFAFFFFF<<FFAFFAFFAF<F7FFFF7F.FFFFF.AFF<FFFFFF)FA)FFF.FFFF7)FFFFAAAFFFFFFFFFFA<FFF AS:i:0 XS:i:-17 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:4:11506:13259:19980 137 chr10 3464571 6 112M = 3464571 0

TTTCCATGTTAGGAGCTAGGTCTGATGACCTCCCCTAACCCTACAGCTACATACCAGCTCTATGAGTAGAATTTAACTTCATATGGGATGTAGGCAGGGTAATAGTTGCGTA

FAFFF)FFFFAFF7FFFFFAFFFFAAF7FAF)FF<F7)77FFFFFFFAFFFFFF7)AFFFA.FF)F7A<F<FF)77)FFF<FFF.<FFFFFAFFF.AFFFFA.F<FAA)F77 AS:i:-14 XS:i:-20 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:31A29T11A2A25A5A3 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:2:12307:22275:8351 137 chr10 3464726 12 115M = 3464726 0

TTCCAGGAAGCCCCTGCTGCCTCCCCCTGGAAACCCTGCCTGCCGTGAGAAGTCTCCAGGTGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGAGCTTTGAACAT

7FFFAAFFFFF7)FFFFFFF7FAF.<7F7.<FFF<FF7<FFAA.)F.FA<FAFFFFFFF<FFFFFFFFFFFFFF.FAFA<FFF<FF.A7<FF<.FFFAA)F.F).AFF7AF.)FF AS:i:-6 XS:i:-16 XN:i:4 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:0N0N0N0N99T11 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:1:12208:21960:17118 137 chr10 3464736 6 116M = 3464736 0

CCCCTGCTGCCTCCCCCTGGAAACCCTGCCTGCCGTGAGAAGTCTCCAGGTGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGTGCTTTGAACATATTTTATTTTT

FF<FAFFFFFFFFFFFFFFFFF.FFFFFFFFFFFFAFFFFFFFFFFFAFFFFFFFFFFFFFF...FFFFFF.FFFFFFFFAFF<<7FFAAAFFFFFFFF<F.F<FFFFFFFAFFFF AS:i:0 XS:i:-5 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:2:H3HYJBGXX:1:13312:17008:11028 137 chr10 3464737 6 96M = 3464737 0

CCCTGCTGCCTCCCCCTGGAAACCCTGCCTGCCGTGAGAAGTCTCCAGGTGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGTGCT

FFFFFFFFFFFFFFFFFFFFF.FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFAFFFF AS:i:0 XS:i:-5 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:96YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:4:23501:23438:14742 137 chr10 3464759 6 116M = 3464759 0

CCCTGCCTGCCGTGAGAAGTCTCCAGGTGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGTGCTTTGAACATATTTTATTTTTTAAAAGAACTATGTTTGTGAATT

F)FFFFF<FAFFFFFFF.FFFFFFFFFFFFFFFFFFF7AAFFFFFAF.<FFFFFFFF)FFF)AFFFF<..)FFFAFFAAFFFFF<FFF.FFFFFAF7A.<FFAFFA7.<F.FAFF< AS:i:0 XS:i:-5 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:4:22608:22580:1379 137 chr10 3464786 1 115M = 3464786 0

TGGCCCTGTCTGAAAACAGGGACCAGGCAATTTATATCAAGGGTGCTTTGAACATATTTTATTTTTTAAAAGAACTATGTTTGTGAATTTTCCGTATACTGGCAAGCTTTTGAAA

FFAFFFFAFFFFFFFFFFFFFFFFFF.FFFFFFAFAFA7F.7AFFFFFFFFFFAF.FFFFAFFFFFF.A.<AFFFFAAFFF<F)7FFFF<AF.7F.FAFAF7<).FFFFAA<<.< AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:1:21110:15851:11639 137 chr10 3464825 1 116M = 3464825 0

AGGGTGCTTTGAACATATTTTATTTTTTAAAAGAACTATGTTTGTGAATTTTCCGTATACTGGCAAGCTTTTGAAAATGTATTTAATTTTGTAATGTTTACCAATGATTTATTTAC

FFFFFFFFFFFAAFFFFFFAF.FFFFFFFFFFFAFFFAFFFFF.F<AFFFFAFFFFAFFFFFFF7FAFFF<FFAFFFFFFFFFA7FFFF<.A<FFFFFFFFF.<FF.FFFAF<AAA AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:Ppp1r14c

NS500688:2:H3HYJBGXX:4:13605:2329:17680 137 chr10 3464850 1 96M = 3464850 0

TTTAAAAGAACTATGTTTGTGAATTTTCCGTATACTGGCAAGCTTTTGAAAATGTATTTAATTTTGTAATGTTTACCAATGATTTATTTACAAGCT

FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFAFFFFF<FFFFFAFFFFFFFFFFF.FFFFFFFFFFFFF.F.FA AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:96 YT:Z:UPXF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:3:21611:10784:7699 137 chr10 3464862 1 114M = 3464862 0

ATGTTTGTGAATTTTCCGTATACTGGCAAGCTTTTGAAAATGTATTTAATTTTGTAATGTTTACCAATGATTTATTTACAAGCTATTTACTCAAATAAAGGGAGCTGCTTACAA

FFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFF<AFFFFFFFFFFFFFAAFFFFFFFFFFF.FFFFFFFFFFFFF.FFFFFFFFFFFF.FFF<FAFF<FAFAFFFAFFAF AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:2:23204:9837:15537 137 chr10 3464915 1 61M = 3464915 0 GTAATGTTTACCAATGATTTATTTACAAGCTATTTACTCAAATAAAGGGAGCTGCTTAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFAAFFAFFFFFFFFFFFFFAAFF AS:i:-10 XS:i:-10 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:58C1A0 YT:Z:UP XF:Z:Ppp1r14c

NS500688:7:H5FYJBGXX:3:22403:6146:13471 137 chr10 3555451 42 116M = 3555451 0

AGTTTTCTCTGAGTTTGCTTTCCTTCCCTCCACCTGTGATAAGTATGGTCCCTGGGGCTTTACTGAAACGGGGTTTCTTTTAATGCTATTGTGGCTTTGGTAGGGAATGGGAACCT

FFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFAFFFFFFFFFFFFFFFFFF7FFFF.FFFFF<FFFFFFFFFFF7FFFFFFF.<FFFFAFFFFF<FFFFFFFFFFFFFFFFFF<FFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:116 YT:Z:UP XF:Z:__no_feature

NS500688:2:H3HYJBGXX:1:12302:2073:2501 137 chr10 3724572 42 96M = 3724572 0

GCAGAGCATGCTCCAGCTGCTGTGCTGAGCCACTGAATTAATGGTACTATGTGGTGCAGTGATAGACAAACAACCACAGCAAGCAAGTGGACTCTC

FFFF7FFFFFFFFFFAFFFFFFFFFFFAFFFFFFFFFFF7FFFFF7FFFFFFFFFFFFFFFFFFF7FFA)F.)FFFFFFFFF<FF7FFFF<FFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:96 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:4:12402:13489:9837 137 chr10 3741923 42 115M = 3741923 0

TTCCTGACTTAGCTTTTTGTCTCTTGGAATTGCACCCCGTCTAAGCCGTTAAAACCGACCAGGAAGAAACTTTCTTTTCCAGAGTTCACTGACCTCCCTGGCTTCATCCGCAAGT

FFFFFAFFAFFFFFFFFA<FFFFFFA.FFFFFF7F7FFFFFFFAF7FFF.FFFF)FFAFAAF.<).FFFFFFFFF)F7.FFF.F)7F.FFFF.FFF7FFA.FFFF...<.AF<FF AS:i:-3 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:105C9 YT:Z:UP XF:Z:__no_feature

On Mon, Apr 27, 2015 at 11:30 PM, Li Shen notifications@github.com wrote:

I'm not sure what is going on. Can you show us your bam's head?

Li

On Mon, Apr 27, 2015 at 10:43 AM, orzechoj notifications@github.com wrote:

Hi Shen Lab,

I have a similar problem: Running ngs.plot.r produces plots where all values are 0. I run the following command

ngs.plot.r -G mm10 -R genebody -C my.bam -O outPrefix

(I installed the mm10 genome, and I didn't get any problems there.) When I run the program on the supplied example

ngs.plot.r -G hg19 -R tss -C hesc.H3k4me3.1M.bam -O hesc.H3k4me3.tss -T H3K4me3 -L 3000 -FL 300

everything worked ok.

I checked that the chromosome names in the mm10 genome.coord object are the same as in the bam file, e.g. "chr12", so for me that's not the problem. I also tried "-R tss", and/or "-D refseq" and got similar results. Also, "-MQ 1", "-F rnaseq" and "-SS same" didn't change anything.

(I ran this using ngsplot-2.47 as well as the ngsplot-develop version downloaded from gitHub 4/26-2015.)

Do you have any ideas what I could try next?

kind regards /Jakub

— Reply to this email directly or view it on GitHub <

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Li Shen, Ph.D. Computer Science Assistant Professor Department of Neuroscience Icahn School of Medicine at Mount Sinai New York, NY 10029 ################# ngs.plot - the coolest NGS plotting tool: https://code.google.com/p/ngsplot/ Want ChIP-seq differential analysis? Use: https://code.google.com/p/diffreps/

— Reply to this email directly or view it on GitHub < https://github.com/shenlab-sinai/ngsplot/issues/39#issuecomment-96827588> .

— Reply to this email directly or view it on GitHub https://github.com/shenlab-sinai/ngsplot/issues/39#issuecomment-96951569 .

Li Shen, Ph.D. Computer Science Assistant Professor Department of Neuroscience Icahn School of Medicine at Mount Sinai New York, NY 10029 ################# ngs.plot - the coolest NGS plotting tool: https://code.google.com/p/ngsplot/ Want ChIP-seq differential analysis? Use: https://code.google.com/p/diffreps/

orzechoj commented 9 years ago

Thanks, will try that. /Jakub

On Wednesday, April 29, 2015, Li Shen notifications@github.com wrote:

I think I found the reason. The SAM flags in your bam file are either 137 or 153, which means "read paired", "mate unmapped", "second in pair" and "read reverse strand" (optional). Because ngs.plot understands paired-end alignments and sees these reads as inappropriate, they are all filtered.

What you need to do is to modify the SAM flags to fool the program into believing that they are single-end alignments. An option is to use bedtools to convert your bam into bed and then convert it back into bam. During this conversion, the unnecessary flags will be discarded.

Li

On Tue, Apr 28, 2015 at 3:03 AM, orzechoj notifications@github.com wrote:

Hi Li, thank you for the quick reply,

The bam file I'm using is a bit weird. The library was generated using a special protocol, so what I have is paired end data, but only the second read in each pair is used. The data were mapped with bowtie2. Below are the first 10 lines, apologies for the lousy formatting.

best, Jakub

$ samtools view R1_formated_mapped_filtered_annotated_SMALL_SORT.bam | head -n 10

NS500688:7:H5FYJBGXX:2:13110:2209:5781 153 chr10 3139861 0 76M1I37M = 3139861 0

ATATTTGGAGATAAATTAGTTGGTCCATAGAGTCTTATCTGGGGAAGACAGGTCCGATAAGCCTCTACAATGCCTTGGATTCCATTGCAATAGGGGTTCGATGGGTTAAAGCTG

7FA)FFFFAAF)F.F7))<)<.7)FF..AFF<)FF<FAA7FFFFFFAF.FFFFA<FF7F<FFA<F<FAFFFFFFFF<FFFFFFFFFFFFFFFFFFF.AFFFFFFF7FFFFFFFF AS:i:-53 XN:i:0 XM:i:11 XO:i:1 XG:i:1 NM:i:12

MD:Z:3A7A10A4C13A12T12T32G9T0C0C0 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:22103:17410:17927 153 chr10 3140296 8 5M1I90M = 3140296 0

GTCTCTTTTTTTTCATTTATGCATTCATATTCAACAGGTGAGCTTCTGGAGGCTTCTTTGAGTTTTGTCATGGTGGTTTGAAATGTTCCAATGAGA

FFFF<FFFFFFFFAFFAFFFFFFAFFFFFFFFAFFFFA<FFFF<.FFFFFFFF)7FFFFFFF.AFFF<FFFFF.FF))FFFFFFFAFFFFFFFAFF AS:i:-33 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:24A9T1A4T40A12 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:22510:19317:1639 153 chr10 3140491 3 23M1D93M = 3140491 0

GCTTCCACATGGGATTCAGGTTGTTTTTTATAACCTCTGTTCGGTGAACCATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGA

FA<<FF7<F7FFFF).FFFF7FF<F7AFAFFFAFFAFFFAAFFFAFFFFFFFFFF<<FFAFFFFFFFFFFFFFFFFF<FF<FFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFF AS:i:-42 XN:i:0 XM:i:7 XO:i:1 XG:i:1 NM:i:8 MD:Z:6C1G14^T11T0C6A20T0C51 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:13206:16212:9184 153 chr10 3140541 24 116M = 3140541 0

CATCAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGT

FF<FFFFFFF7F<FFFFFFFFFFAFFFF<<FFFFF<FF<FFFFFFFF<FFFFFFFAFFFFFAFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-25 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:14T0C77T1C4G15 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:13504:14353:9068 153 chr10 3140544 23 116M = 3140544 0

CAGCCAGTGGCCATCTGATGTCTGCTTGTGAAACTCCAAGTATGGATCTGACTTCCCAAACAGATCCTTATTATCCAGTTTTCTGGCTTCCATTTCAAACAATACCACTCTGTTAT

FFFFAFF..FFFF.AFFF.F.F.FFAAF.FFFFFFFFFA<7A7)AF)FFFFF<FFFF7A<FFFFAFFFFFAFFFFFFF..F7F<FFFAAFFFA.FFFAAFFAFFFFFF.F<F7AFF AS:i:-30 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:11T0C77T1C4G16G1 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:3:23503:14378:11418 153 chr10 3140637 0 115M = 3140637 0

TTCAAACAATACCACTCTGTTATCCTTTATTTCTTCTGCTGAAATCGTAATACTCCCTTTCCCTGCAGGTTTGCCATTTTTGAGCACCAGTGGTCGAGTTAGCTTCTTACTGGAA

AFFFA7FF<<FFFFFFF7FFFFAFFAF.FFFFFAFF7FFFFFFFFAFAF<FFFAF.FFAFFFFFFFFFFF.FFFFFFF<7FFFFFAF7FFFFFAAFFFFFFFAFFFFFFFFFFFA AS:i:-65 XN:i:0 XM:i:13 XO:i:0 XG:i:0 NM:i:13 MD:Z:4G16G15A7T1C3G6C6A26A1T0A5T10T2 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:2:23310:16699:2534 153 chr10 3361500 42 115M = 3361500 0

TTTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA

FFAAAF7F7<FFA.FFF<FF.F<.7FFFFFFFF.FFFFAFFFFAAFFFFFFFFFFFFFFFAFFAFFFFFFAFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:11207:15928:8706 153 chr10 3361501 42 114M = 3361501 0

TTTTAAATTATTTGAAAAACACTGGGTTAGAATCTTTTTACATTGCTGAGAATATCTGGCCCCCAACATTCACTATACGGTTTTTGTTTTTGTTCTATTAATAAATTGATAAGA

AAFAFAFFFFFFAFFAFFFAAFFF<F.FFFFF.FFFFFFFFFF<FFFFFFFFFFAFFFFFFFFFFFFFFFFFF7FFFFFF<FFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YT:Z:UP XF:Z:__no_feature

NS500688:7:H5FYJBGXX:1:12108:17217:19 > https://github.com/shenlab-sinai/ngsplot/issues/39#issuecomment-96827588>

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Li Shen, Ph.D. Computer Science Assistant Professor Department of Neuroscience Icahn School of Medicine at Mount Sinai New York, NY 10029 ################# ngs.plot - the coolest NGS plotting tool: https://code.google.com/p/ngsplot/ Want ChIP-seq differential analysis? Use: https://code.google.com/p/diffreps/

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mforoozani1 commented 7 years ago

@GoogleCodeExporter I have the same issue, I correct the chr in the header of bam file, and as you said I remake the bai file, but I still got the same error. Do you have any idea? this is the command used to modified the bam file:

lishen commented 7 years ago

You shall also check the chromosome names in ngs plot database to ensure consistency.

Sent from my iPhone

On Jul 18, 2017, at 5:09 PM, Maryam notifications@github.com wrote:

@GoogleCodeExporter I have the same issue, I correct the chr in the header of bam file, and as you said I remake the bai file, but I still got the same error. Do you have any idea? this is the command used to modified the bam file:

samtools view -H Tair10_SRR1964977.bam > header_Tair10_SRR1964977.bam sed -e 's/SN:Chr/SN:chr/' header_Tair10_SRR1964977.bam > header_corrected_Tair10_SRR1964977.bam samtools reheader header_corrected_Tair10_SRR1964977.bam Tair10_SRR1964977.bam > modified_Tair10_SRR1964977.bam samtools sort modified_Tair10_SRR1964977.bam sorted_modified_Tair10_SRR1964977.bam samtools index sorted_modified_Tair10_SRR1964977.bam — You are receiving this because you commented. Reply to this email directly, view it on GitHub, or mute the thread.