Analysis of whole genome of an individual (HG02759) from the 1000 genome project

A1. References and resources

We will analyze the HG02759 low coverage genome sequencing data.

1000_genome_HG02759

Machine

Google Cloud Platform (GCP), 4 vCPUs, 15 GB

Google Cloud Platform (GCP), 24 vCPUs, 32 GB

A2. Download analysis datasets (the fastq files)

~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251295/ERR251295_2.fastq.gz

Continuing in background, pid 25770.

~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251295/ERR251295_1.fastq.gz

Continuing in background, pid 25776.

~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251296/ERR251296_2.fastq.gz

Continuing in background, pid 25788.

~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251296/ERR251296_1.fastq.gz

Continuing in background, pid 25790.

Directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 5595686029 Apr 22 02:03 ERR251295_1.fastq.gz

-rw-rw-r– 1 b0d2647 b0d2647 5719449352 Apr 22 02:03 ERR251295_2.fastq.gz

-rw-rw-r– 1 b0d2647 b0d2647 5653494965 Apr 21 22:30 ERR251296_1.fastq.gz

-rw-rw-r– 1 b0d2647 b0d2647 5772390726 Apr 21 22:19 ERR251296_2.fastq.gz

A3. Dwonload the refrence and/or genome files (from the GATK resources bundle maintained by Broad Institure in a google bucket)

~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta ./

Copying gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta…

~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.dict ./

Copying gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.dict…

~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta.fai ./

Copying gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta.fai…

Additional directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 581712 Apr 20 13:42 Homo_sapiens_assembly38.dict

-rw-rw-r– 1 b0d2647 b0d2647 3249912778 Apr 20 13:44 Homo_sapiens_assembly38.fasta

-rw-rw-r– 1 b0d2647 b0d2647 160928 Apr 20 16:47 Homo_sapiens_assembly38.fasta.fai

A4. Make a BWA index of the human genome

~/gatk_data_ref$ bwa index -a bwtsw Homo_sapiens_assembly38.fasta

[bwa_index] Pack FASTA… 30.70 sec

[bwa_index] Construct BWT for the packed sequence…

[BWTIncCreate] textLength=6434693834, availableWord=464768632

[BWTIncConstructFromPacked] 10 iterations done. 99999994 characters processed.

[BWTIncConstructFromPacked] 20 iterations done. 199999994 characters processed.

………….

[BWTIncConstructFromPacked] 700 iterations done. 6411146922 characters processed.

[BWTIncConstructFromPacked] 710 iterations done. 6432978554 characters processed.

[bwt_gen] Finished constructing BWT in 711 iterations.

[bwa_index] 4734.94 seconds elapse.

[bwa_index] Update BWT… 23.36 sec

[bwa_index] Pack forward-only FASTA… 22.12 sec

[bwa_index] Construct SA from BWT and Occ… 2665.77 sec

[main] Version: 0.7.17-r1188

[main] CMD: bwa index -a bwtsw Homo_sapiens_assembly38.fasta

[main] Real time: 7558.780 sec; CPU: 7476.896 sec

Additional directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 487553 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.alt

-rw-rw-r– 1 b0d2647 b0d2647 20199 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.amb

-rw-rw-r– 1 b0d2647 b0d2647 455474 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.ann

-rw-rw-r– 1 b0d2647 b0d2647 3217347004 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.bwt

-rw-rw-r– 1 b0d2647 b0d2647 804336731 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.pac

-rw-rw-r– 1 b0d2647 b0d2647 1608673512 Apr 21 22:50 Homo_sapiens_assembly38.fasta.64.sa

A5. Align the 1st pair of sequences to the reference genome using Burrows-Wheeler Aligner (BWA)

Note: It is important to assign read groups (RG) to the sequences

Check the fastq file contents

~/gatk_data_ref$ zcat ERR251295_1.fastq.gz | head

@ERR251295.1 FCC1H7WACXX:1:1101:1133:2181/1 TGCTNAAAATACAGAAGACAAATTTTCTCATTTAGGGAAATCAATTTTATTAGTGCTTGGTCAGAAAAGACTAAAAATTTCATGTTGAATACGTACTAAT + CCCF#2ADHHHHGIGGHGIIIGHIJJJIIHEIIGGIJJJJIJJCGGHIIHJHIGHIEIJJBGIIIJJGIGIIIJIEEEEHFFFFCDEEEEEEDCCBBEDD

@ERR251295.2 FCC1H7WACXX:1:1101:1086:2200/1 TGACNATTCCATTCAATTCTGTTCAATGATTCCCTTAGATTCCATTTGATGATGATTCCATTCGATTCCATTTGATGATGATTCCATGCGATTCCATTAG + BC@F#2ADDHHHHJJIHIIJIJJJJJJIGIJJJJJJGHIJJIJBHIJIGHIFIIHIJJIJIJJJIJIIH>DHIIHEGHHGGHGJIJJJJJIGHHHHHFFF

@ERR251295.3 FCC1H7WACXX:1:1101:1094:2225/1 GCTGNGAGCCTCGTTGCCATGTAGGCAACTGTATTGAAAGAGTCCAGGCTCCTCCTCAGATTAGGGACTCATTTCCTCTCCCCTCTACAAGCCCGGTGTT

~/gatk_data_ref$ zcat ERR251295_2.fastq.gz | head -n 2

@ERR251295.1 FCC1H7WACXX:1:1101:1133:2181/2 TTGTCTAGTTACTTGAACAATAAGAGAAATTCTATCTTTGAGCTTTCATAATTGGCTTTCTGGACACCACTGAGTTTATTAGATAATCTTTAAAAATATT

~/gatk-data-ref$ bwa mem -M -t 24 -R ‘@RG:ERR251295:Z:illumina:FCC1H7WACXX:HG02759’ Homo_sapiens_assembly38.fasta ERR251295_1.fastq.gz ERR251295_2.fastq.gz | samtools view -Sbh | samtools sort -o ERR251295_1.bam - && samtools index ERR251295_1.bam && samtools flagstat ERR251295_1.bam

…………….

[main] Version: 0.7.17-r1188

[main] Real time: 5776.739 sec; CPU: 81456.772 sec

[bam_sort_core] merging from 49 files and 1 in-memory blocks…

129330360 + 0 in total (QC-passed reads + QC-failed reads)

1128310 + 0 secondary

832800 + 0 supplementary

0 + 0 duplicates

128944247 + 0 mapped (99.70% : N/A)

127369250 + 0 paired in sequencing

63684625 + 0 read1

63684625 + 0 read2

122823900 + 0 properly paired (96.43% : N/A)

126601922 + 0 with itself and mate mapped

381215 + 0 singletons (0.30% : N/A)

2253238 + 0 with mate mapped to a different chr

1052000 + 0 with mate mapped to a different chr (mapQ>=5)

Additional directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 12241966896 Apr 26 22:08 ERR251295_1.bam

-rw-rw-r– 1 b0d2647 b0d2647 6665728 Apr 26 22:12 ERR251295_1.bam.bai

A6. Validate the BAM file and fix it if necessary

~/gatk-data-ref$ gatk ValidateSamFile -R Homo_sapiens_assembly38.fasta -I ERR251295_1.bam -M SUMMARY -O summary_ERR251295_1

Using GATK jar /home/b0d2647/miniconda3/share/gatk4-4.1.2.0-0/gatk-package-4.1.2.0-local.jar

…………………

INFO 2019-04-27 23:39:51 SamFileValidator Validated Read 10,000,000 records. Elapsed time: 00:01:41s. Time for last 10,000,000: 100s. Last read position: chr1:221,207,964

INFO 2019-04-27 23:41:24 SamFileValidator Validated Read 20,000,000 records. Elapsed time: 00:03:14s. Time for last 10,000,000:93s. Last read position: chr2:211,520,747

…………………….

INFO 2019-04-27 23:58:03 SamFileValidator Validated Read 120,000,000 records. Elapsed time: 00:19:53s. Time for last 10,000,000: 104s. Last read position: chr22:47,204,986

[Sun Apr 28 00:06:18 UTC 2019] picard.sam.ValidateSamFile done. Elapsed time: 28.17 minutes.

To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp

Tool returned: 3

Additional directory contents

~/gatk-data-ref$ cat summary_ERR251295_1

## HISTOGRAM    java.lang.String

Error   Type              Count

ERROR:  INVALID_TAG_NM    16816

A7. Fix the error in the BAM file using samtools

~/gatk-data-ref$ samtools calmd -bAr ERR251295_1.bam Homo_sapiens_assembly38.fasta > ERR251295_1.calmd.bam

….

[bam_fillmd1] different MD for read ‘ERR251295.60210117’: ‘33G0T19C0C1C4C11C23’ -> ‘0N32G0T19C0C1C4C11C23’

[bam_fillmd1] different NM for read ‘ERR251295.59668755’: 0 -> 4

[bam_fillmd1] different MD for read ‘ERR251295.59668755’: ‘54’ -> ‘0N0N0N0N50’

[bam_fillmd1] different NM for read ‘ERR251295.47869477’: 0 -> 1

[bam_fillmd1] different MD for read ‘ERR251295.47869477’: ‘73’ -> ‘72N0’

Additional directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 13319762424 Apr 28 01:54 ERR251295_1.calmd.bam

A8. Mark duplicates

~/gatk-data-ref$ gatk MarkDuplicates -I ERR251295_1.calmd.bam -O ERR251295_1.calmd.mdup.bam -M ERR251295_1.calmd.dupMetrics.txt

Additional directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 3525 Apr 28 04:53 ERR251295_1.calmd.dupMetrics.txt

-rw-rw-r– 1 b0d2647 b0d2647 17998947047 Apr 28 04:53 ERR251295_1.calmd.mdup.bam

A9. Index the BAM file

~/gatk-data-ref$ samtools index ERR251295_1.calmd.mdup.bam

Additional directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 8635016 Apr 28 05:06 ERR251295_1.calmd.mdup.bam.bai

A10. Run gatk HaplotypeCaller

~/gatk-data-ref$ gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR251295_1.calmd.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR251295_1.calmd.mdup.vcf

05:10:13.535 INFO ProgressMeter - Current Locus Elapsed Minutes Regions Processed Regions/Minute

05:10:23.556 INFO ProgressMeter - chr20:16090633 0.2 1270 7604.8

05:10:23.995 INFO HaplotypeCaller -

    167 read(s) filtered by: ((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND GoodCigarReadFilter) AND WellformedReadFilter)  

    167 read(s) filtered by: (((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND GoodCigarReadFilter)


  167 read(s) filtered by: ((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter)
  
      167 read(s) filtered by: (((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter)
      
          167 read(s) filtered by: ((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter)
          
              63 read(s) filtered by: (((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter)
              
                  56 read(s) filtered by: ((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter)
                  
                      56 read(s) filtered by: (MappingQualityReadFilter AND MappingQualityAvailableReadFilter)
                      
                          56 read(s) filtered by: MappingQualityReadFilter 
                          
                            7 read(s) filtered by: NotSecondaryAlignmentReadFilter 
                  
                                    104 read(s) filtered by: NotDuplicateReadFilter

05:10:24.008 INFO ProgressMeter - chr20:16098307 0.2 1317 7545.1

05:10:24.009 INFO ProgressMeter - Traversal complete. Processed 1317 total regions in 0.2 minutes.

Additional directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 212003 Apr 28 05:10 ERR251295_1.calmd.mdup.vcf

-rw-rw-r– 1 b0d2647 b0d2647 115099 Apr 28 05:10 ERR251295_1.calmd.mdup.vcf.idx

A11. Record short variants (i.e., SNPs and indels) in the intended chromosomal region

~/gatk-data-ref$ bcftools stats ERR251295_1.calmd.mdup.vcf

# This file was produced by bcftools stats (1.9+htslib-1.9) and can be plotted using plot-vcfstats.


# SN    [2]id   [3]key                                  [4]value

SN      0       number of samples:                      1

SN      0       number of records:                      200

SN      0       number of no-ALTs:                      0

SN      0       number of SNPs:                         196

SN      0       number of MNPs:                         0

SN      0       number of indels:                       4

SN      0       number of others:                       0

SN      0       number of multiallelic sites:           0

SN      0       number of multiallelic SNP sites:       0

B1. Align the 2nd pair of sequences to the reference genome using BWA and analyze as above

File contents

~/gatk-data-ref$ zcat ERR251296_1.fastq.gz | head -n 2

@ERR251296.1 FCC1H7WACXX:2:1101:1495:2145/1

NTCAGAAACTTGTTTGTGATGTGTGCCCTCTACTGACTGAGTTGAACCTTTCTTTTCATAGAGCAGTTTTGAAACACTCTTTTTGTAGAATCTGCAAGAG

~/gatk-data-ref$ zcat ERR251296_2.fastq.gz | head -n 2

@ERR251296.1 FCC1H7WACXX:2:1101:1495:2145/2

GCAAATATCCTCTTGCAGATTCCAGAAAAAGAGTGTTTCAAAACTGCTCCTTCAAAACGGTGGTTCAATTCTCTTAGTTGAGTACACACATCTCAAATAA

~/gatk-data-ref$ bwa mem -M -t 24 -R ‘@RG:ERR251296:Z:illumina:FCC1H7WACXX:HG02759’ Homo_sapiens_assembly38.fasta ERR251296_1.fastq.gz ERR251296_2.fastq.gz | samtools view -Sbh | samtools sort -o ERR251296_1.bam - && samtools index ERR251296_1.bam && samtools flagstat ERR251296_1.bam

…………

[main] Version: 0.7.17-r1188

……………………

[main] Real time: 3407.936 sec; CPU: 72679.793 sec

[bam_sort_core] merging from 49 files and 1 in-memory blocks…

129958650 + 0 in total (QC-passed reads + QC-failed reads)

1139152 + 0 secondary

838006 + 0 supplementary

0 + 0 duplicates

129602327 + 0 mapped (99.73% : N/A)

127981492 + 0 paired in sequencing

63990746 + 0 read1

63990746 + 0 read2

123468430 + 0 properly paired (96.47% : N/A)

127273926 + 0 with itself and mate mapped

351243 + 0 singletons (0.27% : N/A)

2269832 + 0 with mate mapped to a different chr

1057580 + 0 with mate mapped to a different chr (mapQ>=5)

B2. Validate BAM file and fix it if necessary

~/gatk-data-ref$ gatk ValidateSamFile -R Homo_sapiens_assembly38.fasta -I ERR251296_1.bam -M SUMMARY -O summary_ERR251296_1

Using GATK jar /home/b0d2647/miniconda3/share/gatk4-4.1.2.0-0/gatk-package-4.1.2.0-local.jar

Tool returned: 3

~/gatk-data-ref$ cat summary_ERR251296_1

## HISTOGRAM    java.lang.String

Error Type              Count

ERROR:INVALID_TAG_NM    17041

B3. Fix the error (INVALID_TAG_NM)

~/gatk-data-ref$ samtools calmd -bAr ERR251296_1.bam Homo_sapiens_assembly38.fasta > ERR251296_1.calmd.bam

B4. Mark duplicates

~/gatk-data-ref$ gatk MarkDuplicates -I ERR251296_1.calmd.bam -O ERR251296_1.calmd.mdup.bam -M ERR251296_1.calmd.dupMetrics.txt

……………..

INFO 2019-04-28 04:31:40 SortingCollection Creating merging iterator from 6 files

INFO 2019-04-28 04:32:30 MarkDuplicates Traversing fragment information and detecting duplicates.

INFO 2019-04-28 04:32:30 SortingCollection Creating merging iterator from 11 files

INFO 2019-04-28 04:33:42 MarkDuplicates Sorting list of duplicate records.

INFO 2019-04-28 04:33:42 MarkDuplicates After generateDuplicateIndexes freeMemory: 2790920768; totalMemory: 3755474944; maxMemory: 3755474944

INFO 2019-04-28 04:33:42 MarkDuplicates Marking 1927552 records as duplicates.

INFO 2019-04-28 04:33:42 MarkDuplicates Found 0 optical duplicate clusters.

INFO 2019-04-28 04:33:43 MarkDuplicates Reads are assumed to be ordered by: coordinate

INFO 2019-04-28 04:35:48 MarkDuplicates Written 10,000,000 records. Elapsed time: 00:02:05s. Time for last 10,000,000:125s. Last read position: chr1:220,296,254

INFO 2019-04-28 04:38:00 MarkDuplicates Written 20,000,000 records. Elapsed time: 00:04:17s. Time for last 10,000,000:131s. Last read position: chr2:209,223,795

………………….

INFO 2019-04-28 04:53:37 MarkDuplicates Written 120,000,000 records. Elapsed time: 00:19:54s. Time for last 10,000,000:87s. Last read position: chr22:35,225,173

………………………

[Sun Apr 28 04:54:47 UTC 2019] picard.sam.markduplicates.MarkDuplicates done. Elapsed time: 50.44 minutes.

Tool returned:0

B5. Index the BAM file

~/gatk-data-ref$ samtools index ERR251296_1.calmd.mdup.bam

B6. Run gatk HaplotypeCaller

~/gatk-data-ref$ gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR251296_1.calmd.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR251296_1.calmd.mdup.vcf

05:12:24.446 INFO HaplotypeCaller - 158 read(s) filtered by: ((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadF ilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentRe adFilter) AND GoodCigarReadFilter) AND WellformedReadFilter)

……………………..

05:12:24.448 INFO ProgressMeter - chr20:16098789 0.1 1317 9246.4

05:12:24.448 INFO ProgressMeter - Traversal complete. Processed 1317 total regions in 0.1 minutes.

B7. Record short variants (SNPs and indels)

~/gatk-data-ref$ bcftools stats ERR251296_1.calmd.mdup.vcf

# This file was produced by bcftools stats (1.9+htslib-1.9) and can be plotted using plot-vcfstats.

.............

# SN    [2]id   [3]key                                  [4]value

SN      0       number of samples:                      1

SN      0       number of records:                      207

SN      0       number of no-ALTs:                      0

SN      0       number of SNPs:                         202

SN      0       number of MNPs:                         0

SN      0       number of indels:                       5

SN      0       number of others:                       0

SN      0       number of multiallelic sites:           0

SN      0       number of multiallelic SNP sites:       0

B8. Additional directory contents as a result of 2nd pair of sequence alignment

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 12359531427 Apr 28 00:39 ERR251296_1.bam

-rw-rw-r– 1 b0d2647 b0d2647 6765576 Apr 28 00:41 ERR251296_1.bam.bai

-rw-rw-r– 1 b0d2647 b0d2647 13441427070 Apr 28 02:59 ERR251296_1.calmd.bam

-rw-rw-r– 1 b0d2647 b0d2647 3531 Apr 28 04:54 ERR251296_1.calmd.dupMetrics.txt

-rw-rw-r– 1 b0d2647 b0d2647 18167621403 Apr 28 04:54 ERR251296_1.calmd.mdup.bam

-rw-rw-r– 1 b0d2647 b0d2647 8673408 Apr 28 05:07 ERR251296_1.calmd.mdup.bam.bai

-rw-rw-r– 1 b0d2647 b0d2647 213523 Apr 28 05:12 ERR251296_1.calmd.mdup.vcf

-rw-rw-r– 1 b0d2647 b0d2647 115099 Apr 28 05:12 ERR251296_1.calmd.mdup.vcf.idx

BAM files could be merged and analyzed for combined analysis of multiple sequencing pairs of the same genome

C1. Merge the two BAM files

~/gatk-data-ref$ gatk MergeSamFiles -I ERR251295_1.calmd.bam -I ERR251296_1.calmd.bam -O ERR25129596.bam

………………….

INFO 2019-04-28 03:50:00 MergeSamFiles Processed 259,000,000 records. Elapsed time: 00:19:20s. Time for last 1,000,000: 7s.Last read position:chrUn_JTFH01001899v1_decoy:273

INFO 2019-04-28 03:50:02 MergeSamFiles Finished reading inputs.

[Sun Apr 28 03:50:02 UTC 2019] picard.sam.MergeSamFiles done. Elapsed time: 19.39 minutes.

C2. Mark duplicates

~/gatk-data-ref$ gatk MarkDuplicates -I ERR25129596.bam -O ERR25129596.mdup.bam -M ERR25129596.dupMetrics.txt

INFO 2019-04-28 04:44:56 SortingCollection Creating merging iterator from 10 files

INFO 2019-04-28 04:46:32 MarkDuplicates Traversing fragment information and detecting duplicates.

INFO 2019-04-28 04:46:32 SortingCollection Creating merging iterator from 21 files

INFO 2019-04-28 04:48:49 MarkDuplicates Sorting list of duplicate records.

INFO 2019-04-28 04:48:51 MarkDuplicates After generateDuplicateIndexes freeMemory: 2927996376; totalMemory: 3935305728; maxMemory: 3935305 728 INFO 2019-04-28 04:48:51 MarkDuplicates Marking 6604590 records as duplicates.

INFO 2019-04-28 04:48:51 MarkDuplicates Found 0 optical duplicate clusters.

INFO 2019-04-28 04:48:51 MarkDuplicates Reads are assumed to be ordered by: coordinate

INFO 2019-04-28 04:50:23 MarkDuplicates Written 10,000,000 records. Elapsed time: 00:01:31s. Time for last 10,000,000: 91s. Last read position: chr1:114,332,129

………………….

INFO 2019-04-28 05:11:03 MarkDuplicates Written 250,000,000 records. Elapsed time: 00:22:11s. Time for last 10,000,000: 49s. Last read position: chrY:56,737,140

……………………

[Sun Apr 28 05:11:50 UTC 2019] picard.sam.markduplicates.MarkDuplicates done. Elapsed time: 76.69 minutes.

C3. Index the BAM file

samtools index ERR25129596.mdup.bam

C4. Run gatk HaplotypeCaller

gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR25129596.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR25129596.mdup.vcf

05:29:40.276 INFO ProgressMeter - Current Locus Elapsed Minutes Regions Processed Regions/Minute

05:29:50.098 INFO HaplotypeCaller -

500 read(s) filtered by: ((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND GoodCigarReadFilter) AND WellformedReadFilter)

500 read(s) filtered by: (((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND GoodCigarReadFilter)

  500 read(s) filtered by: ((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter)
      
      500 read(s) filtered by: (((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter)
          
          500 read(s) filtered by: ((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter)
              
              133 read(s) filtered by: (((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter)
                  
                  122 read(s) filtered by: ((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter)
                      
                      122 read(s) filtered by: (MappingQualityReadFilter AND MappingQualityAvailableReadFilter)
                          
                          122 read(s) filtered by: MappingQualityReadFilter 
                  
                  11 read(s) filtered by: NotSecondaryAlignmentReadFilter 
             
              367 read(s) filtered by: NotDuplicateReadFilter

05:29:50.098 INFO ProgressMeter - chr20:16099540 0.2 1373 8387.3

05:29:50.099 INFO ProgressMeter - Traversal complete. Processed 1373 total regions in 0.2 minutes.

C5. Record short variants (SNPs and indels)

~/gatk-data-ref$ bcftools stats ERR25129596.mdup.vcf

# This file was produced by bcftools stats (1.9+htslib-1.9) and can be plotted using plot-vcfstats.
.............

# SN    [2]id   [3]key                                  [4]value

SN      0       number of samples:                      1

SN      0       number of records:                      286

SN      0       number of no-ALTs:                      0

SN      0       number of SNPs:                         276

SN      0       number of MNPs:                         0

SN      0       number of indels:                       10

SN      0       number of others:                       0

SN      0       number of multiallelic sites:           0

SN      0       number of multiallelic SNP sites:       0

C6. Additional directory contents

~/gatk-data-ref$ ls -l

-rw-rw-r– 1 b0d2647 b0d2647 34307229848 Apr 28 03:50 ERR25129596.bam

-rw-rw-r– 1 b0d2647 b0d2647 3569 Apr 28 05:11 ERR25129596.dupMetrics.txt

-rw-rw-r– 1 b0d2647 b0d2647 34564180275 Apr 28 05:11 ERR25129596.mdup.bam

-rw-rw-r– 1 b0d2647 b0d2647 9185888 Apr 28 05:24 ERR25129596.mdup.bam.bai

-rw-rw-r– 1 b0d2647 b0d2647 230777 Apr 28 05:29 ERR25129596.mdup.vcf

-rw-rw-r– 1 b0d2647 b0d2647 116101 Apr 28 05:29 ERR25129596.mdup.vcf.idx

---
title: "Analysis of whole genome of an individual (HG02759) from the 1000 genome project"
author: "Bhagirathi Dash"
output: html_notebook
---


## A1. References and resources

#### We will analyze the HG02759 low coverage genome sequencing data.

![1000_genome_HG02759](HG02759.jpeg)


#### Machine

Google Cloud Platform (GCP), 4 vCPUs, 15 GB
 
Google Cloud Platform (GCP), 24 vCPUs, 32 GB



## A2. Download analysis datasets (the fastq files)


#### ~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251295/ERR251295_2.fastq.gz

Continuing in background, pid 25770.

#### ~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251295/ERR251295_1.fastq.gz

Continuing in background, pid 25776.

#### ~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251296/ERR251296_2.fastq.gz

Continuing in background, pid 25788.

#### ~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251296/ERR251296_1.fastq.gz

Continuing in background, pid 25790.



### Directory contents

#### ~/gatk-data-ref$ ls -l

-rw-rw-r-- 1 b0d2647 b0d2647  5595686029 Apr 22 02:03 ERR251295_1.fastq.gz

-rw-rw-r-- 1 b0d2647 b0d2647  5719449352 Apr 22 02:03 ERR251295_2.fastq.gz

-rw-rw-r-- 1 b0d2647 b0d2647  5653494965 Apr 21 22:30 ERR251296_1.fastq.gz

-rw-rw-r-- 1 b0d2647 b0d2647  5772390726 Apr 21 22:19 ERR251296_2.fastq.gz




## A3. Dwonload the refrence and/or genome files (from the GATK resources bundle maintained by Broad Institure in a google bucket)



#### ~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta ./

Copying gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta...


#### ~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.dict ./

Copying gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.dict...


#### ~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta.fai ./

Copying gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta.fai...




### Additional directory contents


#### ~/gatk-data-ref$ ls -l

-rw-rw-r-- 1 b0d2647 b0d2647      581712 Apr 20 13:42 Homo_sapiens_assembly38.dict

-rw-rw-r-- 1 b0d2647 b0d2647  3249912778 Apr 20 13:44 Homo_sapiens_assembly38.fasta

-rw-rw-r-- 1 b0d2647 b0d2647      160928 Apr 20 16:47 Homo_sapiens_assembly38.fasta.fai



## A4. Make a BWA index of the human genome





#### ~/gatk_data_ref$ bwa index -a bwtsw Homo_sapiens_assembly38.fasta


[bwa_index] Pack FASTA... 30.70 sec

[bwa_index] Construct BWT for the packed sequence...

[BWTIncCreate] textLength=6434693834, availableWord=464768632

[BWTIncConstructFromPacked] 10 iterations done. 99999994 characters processed.

[BWTIncConstructFromPacked] 20 iterations done. 199999994 characters processed.

.............


[BWTIncConstructFromPacked] 700 iterations done. 6411146922 characters processed.

[BWTIncConstructFromPacked] 710 iterations done. 6432978554 characters processed.

[bwt_gen] Finished constructing BWT in 711 iterations.

[bwa_index] 4734.94 seconds elapse.

[bwa_index] Update BWT... 23.36 sec

[bwa_index] Pack forward-only FASTA... 22.12 sec

[bwa_index] Construct SA from BWT and Occ... 2665.77 sec

[main] Version: 0.7.17-r1188

[main] CMD: bwa index -a bwtsw Homo_sapiens_assembly38.fasta

[main] Real time: 7558.780 sec; CPU: 7476.896 sec


### Additional directory contents

#### ~/gatk-data-ref$ ls -l

-rw-rw-r-- 1 b0d2647 b0d2647      487553 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.alt

-rw-rw-r-- 1 b0d2647 b0d2647       20199 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.amb

-rw-rw-r-- 1 b0d2647 b0d2647      455474 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.ann

-rw-rw-r-- 1 b0d2647 b0d2647  3217347004 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.bwt

-rw-rw-r-- 1 b0d2647 b0d2647   804336731 Apr 21 22:49 Homo_sapiens_assembly38.fasta.64.pac

-rw-rw-r-- 1 b0d2647 b0d2647  1608673512 Apr 21 22:50 Homo_sapiens_assembly38.fasta.64.sa



## A5. Align the 1st pair of sequences to the reference genome using Burrows-Wheeler Aligner (BWA)

Note: It is important to assign read groups (RG) to the sequences

### Check the fastq file contents

####  ~/gatk_data_ref$ zcat ERR251295_1.fastq.gz | head

@ERR251295.1 FCC1H7WACXX:1:1101:1133:2181/1
TGCTNAAAATACAGAAGACAAATTTTCTCATTTAGGGAAATCAATTTTATTAGTGCTTGGTCAGAAAAGACTAAAAATTTCATGTTGAATACGTACTAAT
+
CCCF#2ADHHHHGIGGHGIIIGHIJJJIIHEIIGGIJJJJIJJCGGHIIHJHIGHIEIJJBGIIIJJGIGIIIJIEEEEHFFFFCDEEEEEEDCCBBEDD

@ERR251295.2 FCC1H7WACXX:1:1101:1086:2200/1
TGACNATTCCATTCAATTCTGTTCAATGATTCCCTTAGATTCCATTTGATGATGATTCCATTCGATTCCATTTGATGATGATTCCATGCGATTCCATTAG
+
BC@F#2ADDHHHHJJIHIIJIJJJJJJIGIJJJJJJGHIJJIJBHIJIGHIFIIHIJJIJIJJJIJIIH>DHIIHEGHHGGHGJIJJJJJIGHHHHHFFF

@ERR251295.3 FCC1H7WACXX:1:1101:1094:2225/1
GCTGNGAGCCTCGTTGCCATGTAGGCAACTGTATTGAAAGAGTCCAGGCTCCTCCTCAGATTAGGGACTCATTTCCTCTCCCCTCTACAAGCCCGGTGTT



####  ~/gatk_data_ref$ zcat ERR251295_2.fastq.gz | head -n 2

@ERR251295.1 FCC1H7WACXX:1:1101:1133:2181/2
TTGTCTAGTTACTTGAACAATAAGAGAAATTCTATCTTTGAGCTTTCATAATTGGCTTTCTGGACACCACTGAGTTTATTAGATAATCTTTAAAAATATT


####  ~/gatk-data-ref$ bwa mem -M -t 24 -R '@RG\tID:ERR251295\tLB:Z\tPL:illumina\tPU:FCC1H7WACXX\tSM:HG02759' Homo_sapiens_assembly38.fasta ERR251295_1.fastq.gz ERR251295_2.fastq.gz | samtools view -Sbh | samtools sort -o ERR251295_1.bam - && samtools index ERR251295_1.bam && samtools flagstat ERR251295_1.bam

................

[main] Version: 0.7.17-r1188

[main] Real time: 5776.739 sec; CPU: 81456.772 sec

[bam_sort_core] merging from 49 files and 1 in-memory blocks...


```
129330360 + 0 in total (QC-passed reads + QC-failed reads)

1128310 + 0 secondary

832800 + 0 supplementary

0 + 0 duplicates

128944247 + 0 mapped (99.70% : N/A)

127369250 + 0 paired in sequencing

63684625 + 0 read1

63684625 + 0 read2

122823900 + 0 properly paired (96.43% : N/A)

126601922 + 0 with itself and mate mapped

381215 + 0 singletons (0.30% : N/A)

2253238 + 0 with mate mapped to a different chr

1052000 + 0 with mate mapped to a different chr (mapQ>=5)

```


### Additional directory contents

#### ~/gatk-data-ref$ ls -l

-rw-rw-r-- 1 b0d2647 b0d2647 12241966896 Apr 26 22:08 ERR251295_1.bam

-rw-rw-r-- 1 b0d2647 b0d2647     6665728 Apr 26 22:12 ERR251295_1.bam.bai





## A6. Validate the BAM file and fix it if necessary

####  ~/gatk-data-ref$ gatk ValidateSamFile -R Homo_sapiens_assembly38.fasta -I ERR251295_1.bam -M SUMMARY -O summary_ERR251295_1
 
Using GATK jar /home/b0d2647/miniconda3/share/gatk4-4.1.2.0-0/gatk-package-4.1.2.0-local.jar

.....................

INFO    2019-04-27 23:39:51     SamFileValidator        Validated Read    10,000,000 records.  Elapsed time: 00:01:41s.  Time for last 10,000,000:  100s.  Last read position: chr1:221,207,964


INFO    2019-04-27 23:41:24     SamFileValidator        Validated Read    20,000,000 records.  Elapsed time: 00:03:14s.  Time for last 10,000,000:93s.  Last read position: chr2:211,520,747


.........................

INFO    2019-04-27 23:58:03     SamFileValidator        Validated Read   120,000,000 records.  Elapsed time: 00:19:53s.  Time for last 10,000,000:  104s.  Last read position: chr22:47,204,986


[Sun Apr 28 00:06:18 UTC 2019] picard.sam.ValidateSamFile done. Elapsed time: 28.17 minutes.

To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp

Tool returned: 3


### Additional directory contents


####  ~/gatk-data-ref$ cat summary_ERR251295_1 

```
## HISTOGRAM    java.lang.String

Error   Type              Count

ERROR:  INVALID_TAG_NM    16816
```


## A7. Fix the error in the BAM file using samtools

####  ~/gatk-data-ref$ samtools calmd -bAr ERR251295_1.bam Homo_sapiens_assembly38.fasta > ERR251295_1.calmd.bam

....

[bam_fillmd1] different MD for read 'ERR251295.60210117': '33G0T19C0C1C4C11C23' -> '0N32G0T19C0C1C4C11C23'

[bam_fillmd1] different NM for read 'ERR251295.59668755': 0 -> 4

[bam_fillmd1] different MD for read 'ERR251295.59668755': '54' -> '0N0N0N0N50'

[bam_fillmd1] different NM for read 'ERR251295.47869477': 0 -> 1

[bam_fillmd1] different MD for read 'ERR251295.47869477': '73' -> '72N0'





### Additional directory contents

#### ~/gatk-data-ref$ ls -l





-rw-rw-r-- 1 b0d2647 b0d2647 13319762424 Apr 28 01:54 ERR251295_1.calmd.bam






## A8. Mark duplicates


#### ~/gatk-data-ref$ gatk MarkDuplicates -I ERR251295_1.calmd.bam -O ERR251295_1.calmd.mdup.bam -M ERR251295_1.calmd.dupMetrics.txt




### Additional directory contents

#### ~/gatk-data-ref$ ls -l

-rw-rw-r-- 1 b0d2647 b0d2647        3525 Apr 28 04:53 ERR251295_1.calmd.dupMetrics.txt

-rw-rw-r-- 1 b0d2647 b0d2647 17998947047 Apr 28 04:53 ERR251295_1.calmd.mdup.bam





## A9. Index the BAM file


#### ~/gatk-data-ref$ samtools index ERR251295_1.calmd.mdup.bam


### Additional directory contents

#### ~/gatk-data-ref$ ls -l

-rw-rw-r-- 1 b0d2647 b0d2647     8635016 Apr 28 05:06 ERR251295_1.calmd.mdup.bam.bai



## A10. Run gatk HaplotypeCaller

####  ~/gatk-data-ref$ gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR251295_1.calmd.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR251295_1.calmd.mdup.vcf


05:10:13.535 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute

05:10:23.556 INFO  ProgressMeter -       chr20:16090633              0.2                  1270           7604.8

05:10:23.995 INFO  HaplotypeCaller - 

        167 read(s) filtered by: ((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND GoodCigarReadFilter) AND WellformedReadFilter)  

        167 read(s) filtered by: (((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND GoodCigarReadFilter)


      167 read(s) filtered by: ((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter)
      
          167 read(s) filtered by: (((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter)
          
              167 read(s) filtered by: ((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter)
              
                  63 read(s) filtered by: (((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter)
                  
                      56 read(s) filtered by: ((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter)
                      
                          56 read(s) filtered by: (MappingQualityReadFilter AND MappingQualityAvailableReadFilter)
                          
                              56 read(s) filtered by: MappingQualityReadFilter 
                              
                                7 read(s) filtered by: NotSecondaryAlignmentReadFilter 
                      
                                        104 read(s) filtered by: NotDuplicateReadFilter 
                  
05:10:24.008 INFO  ProgressMeter -       chr20:16098307              0.2                  1317           7545.1

05:10:24.009 INFO  ProgressMeter - Traversal complete. Processed 1317 total regions in 0.2 minutes.




### Additional directory contents

#### ~/gatk-data-ref$ ls -l

-rw-rw-r-- 1 b0d2647 b0d2647      212003 Apr 28 05:10 ERR251295_1.calmd.mdup.vcf

-rw-rw-r-- 1 b0d2647 b0d2647      115099 Apr 28 05:10 ERR251295_1.calmd.mdup.vcf.idx





## A11. Record short variants (i.e., SNPs and indels) in the intended chromosomal region

####  ~/gatk-data-ref$ bcftools stats ERR251295_1.calmd.mdup.vcf

```
# This file was produced by bcftools stats (1.9+htslib-1.9) and can be plotted using plot-vcfstats.


# SN    [2]id   [3]key                                  [4]value

SN      0       number of samples:                      1

SN      0       number of records:                      200

SN      0       number of no-ALTs:                      0

SN      0       number of SNPs:                         196

SN      0       number of MNPs:                         0

SN      0       number of indels:                       4

SN      0       number of others:                       0

SN      0       number of multiallelic sites:           0

SN      0       number of multiallelic SNP sites:       0

```




## B1. Align the 2nd pair of sequences to the reference genome using BWA and analyze as above

### File contents

####  ~/gatk-data-ref$ zcat ERR251296_1.fastq.gz | head -n 2

@ERR251296.1 FCC1H7WACXX:2:1101:1495:2145/1

NTCAGAAACTTGTTTGTGATGTGTGCCCTCTACTGACTGAGTTGAACCTTTCTTTTCATAGAGCAGTTTTGAAACACTCTTTTTGTAGAATCTGCAAGAG

####  ~/gatk-data-ref$ zcat ERR251296_2.fastq.gz | head -n 2

@ERR251296.1 FCC1H7WACXX:2:1101:1495:2145/2

GCAAATATCCTCTTGCAGATTCCAGAAAAAGAGTGTTTCAAAACTGCTCCTTCAAAACGGTGGTTCAATTCTCTTAGTTGAGTACACACATCTCAAATAA


####  ~/gatk-data-ref$ bwa mem -M -t 24 -R '@RG\tID:ERR251296\tLB:Z\tPL:illumina\tPU:FCC1H7WACXX\tSM:HG02759' Homo_sapiens_assembly38.fasta ERR251296_1.fastq.gz ERR251296_2.fastq.gz | samtools view -Sbh | samtools sort -o ERR251296_1.bam - && samtools index ERR251296_1.bam && samtools flagstat ERR251296_1.bam


............

[main] Version: 0.7.17-r1188

........................

[main] Real time: 3407.936 sec; CPU: 72679.793 sec

[bam_sort_core] merging from 49 files and 1 in-memory blocks...


```
129958650 + 0 in total (QC-passed reads + QC-failed reads)

1139152 + 0 secondary

838006 + 0 supplementary

0 + 0 duplicates

129602327 + 0 mapped (99.73% : N/A)

127981492 + 0 paired in sequencing

63990746 + 0 read1

63990746 + 0 read2

123468430 + 0 properly paired (96.47% : N/A)

127273926 + 0 with itself and mate mapped

351243 + 0 singletons (0.27% : N/A)

2269832 + 0 with mate mapped to a different chr

1057580 + 0 with mate mapped to a different chr (mapQ>=5)

```


## B2. Validate BAM file and fix it if necessary


####  ~/gatk-data-ref$ gatk ValidateSamFile -R Homo_sapiens_assembly38.fasta -I ERR251296_1.bam -M SUMMARY -O summary_ERR251296_1

Using GATK jar /home/b0d2647/miniconda3/share/gatk4-4.1.2.0-0/gatk-package-4.1.2.0-local.jar


Tool returned: 3


####  ~/gatk-data-ref$ cat summary_ERR251296_1 

```
## HISTOGRAM    java.lang.String

Error Type              Count

ERROR:INVALID_TAG_NM    17041

```



## B3. Fix the error (INVALID_TAG_NM)

####  ~/gatk-data-ref$ samtools calmd -bAr ERR251296_1.bam Homo_sapiens_assembly38.fasta > ERR251296_1.calmd.bam



## B4. Mark duplicates

####  ~/gatk-data-ref$ gatk MarkDuplicates -I ERR251296_1.calmd.bam -O ERR251296_1.calmd.mdup.bam -M ERR251296_1.calmd.dupMetrics.txt


.................

INFO    2019-04-28 04:31:40     SortingCollection       Creating merging iterator from 6 files

INFO    2019-04-28 04:32:30     MarkDuplicates  Traversing fragment information and detecting duplicates.

INFO    2019-04-28 04:32:30     SortingCollection       Creating merging iterator from 11 files

INFO    2019-04-28 04:33:42     MarkDuplicates  Sorting list of duplicate records.

INFO    2019-04-28 04:33:42     MarkDuplicates  After generateDuplicateIndexes freeMemory: 2790920768; totalMemory: 3755474944; maxMemory: 3755474944

INFO    2019-04-28 04:33:42     MarkDuplicates  Marking 1927552 records as duplicates.

INFO    2019-04-28 04:33:42     MarkDuplicates  Found 0 optical duplicate clusters.

INFO    2019-04-28 04:33:43     MarkDuplicates  Reads are assumed to be ordered by: coordinate

INFO    2019-04-28 04:35:48     MarkDuplicates  Written    10,000,000 records.  Elapsed time: 00:02:05s.  Time for last 10,000,000:125s.  Last read position: chr1:220,296,254
 
INFO    2019-04-28 04:38:00     MarkDuplicates  Written    20,000,000 records.  Elapsed time: 00:04:17s.  Time for last 10,000,000:131s.  Last read position: chr2:209,223,795
 
......................


INFO    2019-04-28 04:53:37     MarkDuplicates  Written   120,000,000 records.  Elapsed time: 00:19:54s.  Time for last 10,000,000:87s.  Last read position: chr22:35,225,173


...........................

[Sun Apr 28 04:54:47 UTC 2019] picard.sam.markduplicates.MarkDuplicates done. Elapsed time: 50.44 minutes.

Tool returned:0




## B5. Index the BAM file

#### ~/gatk-data-ref$ samtools index ERR251296_1.calmd.mdup.bam



## B6. Run gatk HaplotypeCaller



####  ~/gatk-data-ref$ gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR251296_1.calmd.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR251296_1.calmd.mdup.vcf

05:12:24.446 INFO  HaplotypeCaller - 158 read(s) filtered by: ((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadF
ilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentRe
adFilter) AND GoodCigarReadFilter) AND WellformedReadFilter)

..........................
                  
05:12:24.448 INFO  ProgressMeter -       chr20:16098789              0.1                  1317           9246.4

05:12:24.448 INFO  ProgressMeter - Traversal complete. Processed 1317 total regions in 0.1 minutes.




## B7. Record short variants (SNPs and indels)


####  ~/gatk-data-ref$ bcftools stats ERR251296_1.calmd.mdup.vcf


```
# This file was produced by bcftools stats (1.9+htslib-1.9) and can be plotted using plot-vcfstats.

.............

# SN    [2]id   [3]key                                  [4]value

SN      0       number of samples:                      1

SN      0       number of records:                      207

SN      0       number of no-ALTs:                      0

SN      0       number of SNPs:                         202

SN      0       number of MNPs:                         0

SN      0       number of indels:                       5

SN      0       number of others:                       0

SN      0       number of multiallelic sites:           0

SN      0       number of multiallelic SNP sites:       0

```


## B8. Additional directory contents as a result of 2nd pair of sequence alignment

#### ~/gatk-data-ref$ ls -l

-rw-rw-r-- 1 b0d2647 b0d2647 12359531427 Apr 28 00:39 ERR251296_1.bam

-rw-rw-r-- 1 b0d2647 b0d2647     6765576 Apr 28 00:41 ERR251296_1.bam.bai

-rw-rw-r-- 1 b0d2647 b0d2647 13441427070 Apr 28 02:59 ERR251296_1.calmd.bam

-rw-rw-r-- 1 b0d2647 b0d2647        3531 Apr 28 04:54 ERR251296_1.calmd.dupMetrics.txt

-rw-rw-r-- 1 b0d2647 b0d2647 18167621403 Apr 28 04:54 ERR251296_1.calmd.mdup.bam

-rw-rw-r-- 1 b0d2647 b0d2647     8673408 Apr 28 05:07 ERR251296_1.calmd.mdup.bam.bai

-rw-rw-r-- 1 b0d2647 b0d2647      213523 Apr 28 05:12 ERR251296_1.calmd.mdup.vcf

-rw-rw-r-- 1 b0d2647 b0d2647      115099 Apr 28 05:12 ERR251296_1.calmd.mdup.vcf.idx




## BAM files could be merged and analyzed for combined analysis of multiple sequencing pairs of the same genome  



## C1. Merge the two BAM files

####  ~/gatk-data-ref$ gatk MergeSamFiles -I ERR251295_1.calmd.bam -I ERR251296_1.calmd.bam -O ERR25129596.bam

......................
 
 
INFO    2019-04-28 03:50:00     MergeSamFiles   Processed   259,000,000 records.  Elapsed time: 00:19:20s.  Time for last 1,000,000:    7s.Last read position:chrUn_JTFH01001899v1_decoy:273

INFO    2019-04-28 03:50:02     MergeSamFiles   Finished reading inputs.

[Sun Apr 28 03:50:02 UTC 2019] picard.sam.MergeSamFiles done. Elapsed time: 19.39 minutes.



## C2. Mark duplicates


####  ~/gatk-data-ref$ gatk MarkDuplicates -I ERR25129596.bam -O ERR25129596.mdup.bam -M ERR25129596.dupMetrics.txt

INFO    2019-04-28 04:44:56     SortingCollection       Creating merging iterator from 10 files

INFO    2019-04-28 04:46:32     MarkDuplicates  Traversing fragment information and detecting duplicates.

INFO    2019-04-28 04:46:32     SortingCollection       Creating merging iterator from 21 files

INFO    2019-04-28 04:48:49     MarkDuplicates  Sorting list of duplicate records.

INFO    2019-04-28 04:48:51     MarkDuplicates  After generateDuplicateIndexes freeMemory: 2927996376; totalMemory: 3935305728; maxMemory: 3935305
728
INFO    2019-04-28 04:48:51     MarkDuplicates  Marking 6604590 records as duplicates.

INFO    2019-04-28 04:48:51     MarkDuplicates  Found 0 optical duplicate clusters.

INFO    2019-04-28 04:48:51     MarkDuplicates  Reads are assumed to be ordered by: coordinate

INFO    2019-04-28 04:50:23     MarkDuplicates  Written    10,000,000 records.  Elapsed time: 00:01:31s.  Time for last 10,000,000:   91s.  Last read position: chr1:114,332,129

......................

INFO    2019-04-28 05:11:03     MarkDuplicates  Written   250,000,000 records.  Elapsed time: 00:22:11s.  Time for last 10,000,000:   49s.  Last read position: chrY:56,737,140

........................

[Sun Apr 28 05:11:50 UTC 2019] picard.sam.markduplicates.MarkDuplicates done. Elapsed time: 76.69 minutes.





## C3. Index the BAM file


#### samtools index ERR25129596.mdup.bam



## C4. Run gatk HaplotypeCaller


#### gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR25129596.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR25129596.mdup.vcf

05:29:40.276 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute

05:29:50.098 INFO  HaplotypeCaller - 

500 read(s) filtered by: ((((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND GoodCigarReadFilter) AND WellformedReadFilter)

  500 read(s) filtered by: (((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter) AND GoodCigarReadFilter)

      500 read(s) filtered by: ((((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter) AND NonZeroReferenceLengthAlignmentReadFilter)
          
          500 read(s) filtered by: (((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter) AND PassesVendorQualityCheckReadFilter)
              
              500 read(s) filtered by: ((((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter) AND NotDuplicateReadFilter)
                  
                  133 read(s) filtered by: (((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter) AND NotSecondaryAlignmentReadFilter)
                      
                      122 read(s) filtered by: ((MappingQualityReadFilter AND MappingQualityAvailableReadFilter) AND MappedReadFilter)
                          
                          122 read(s) filtered by: (MappingQualityReadFilter AND MappingQualityAvailableReadFilter)
                              
                              122 read(s) filtered by: MappingQualityReadFilter 
                      
                      11 read(s) filtered by: NotSecondaryAlignmentReadFilter 
                 
                  367 read(s) filtered by: NotDuplicateReadFilter 
                  
05:29:50.098 INFO  ProgressMeter -       chr20:16099540              0.2                  1373           8387.3

05:29:50.099 INFO  ProgressMeter - Traversal complete. Processed 1373 total regions in 0.2 minutes.



## C5. Record short variants (SNPs and indels)



####  ~/gatk-data-ref$ bcftools stats ERR25129596.mdup.vcf


```
# This file was produced by bcftools stats (1.9+htslib-1.9) and can be plotted using plot-vcfstats.
.............

# SN    [2]id   [3]key                                  [4]value

SN      0       number of samples:                      1

SN      0       number of records:                      286

SN      0       number of no-ALTs:                      0

SN      0       number of SNPs:                         276

SN      0       number of MNPs:                         0

SN      0       number of indels:                       10

SN      0       number of others:                       0

SN      0       number of multiallelic sites:           0

SN      0       number of multiallelic SNP sites:       0

```

## C6. Additional directory contents

####  ~/gatk-data-ref$ ls -l



-rw-rw-r-- 1 b0d2647 b0d2647 34307229848 Apr 28 03:50 ERR25129596.bam

-rw-rw-r-- 1 b0d2647 b0d2647        3569 Apr 28 05:11 ERR25129596.dupMetrics.txt

-rw-rw-r-- 1 b0d2647 b0d2647 34564180275 Apr 28 05:11 ERR25129596.mdup.bam

-rw-rw-r-- 1 b0d2647 b0d2647     9185888 Apr 28 05:24 ERR25129596.mdup.bam.bai

-rw-rw-r-- 1 b0d2647 b0d2647      230777 Apr 28 05:29 ERR25129596.mdup.vcf

-rw-rw-r-- 1 b0d2647 b0d2647      116101 Apr 28 05:29 ERR25129596.mdup.vcf.idx




Analysis of whole genome of an individual (HG02759) from the 1000 genome project

Bhagirathi Dash

A1. References and resources

We will analyze the HG02759 low coverage genome sequencing data.

Machine

A2. Download analysis datasets (the fastq files)

~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251295/ERR251295_2.fastq.gz

~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251295/ERR251295_1.fastq.gz

~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251296/ERR251296_2.fastq.gz

~/gatk_data_ref$ wget -bqc ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR251/ERR251296/ERR251296_1.fastq.gz

Directory contents

~/gatk-data-ref$ ls -l

A3. Dwonload the refrence and/or genome files (from the GATK resources bundle maintained by Broad Institure in a google bucket)

~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta ./

~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.dict ./

~/gatk_data_ref$ gsutil cp gs://genomics-public-data/resources/broad/hg38/v0/Homo_sapiens_assembly38.fasta.fai ./

Additional directory contents

~/gatk-data-ref$ ls -l

A4. Make a BWA index of the human genome

~/gatk_data_ref$ bwa index -a bwtsw Homo_sapiens_assembly38.fasta

Additional directory contents

~/gatk-data-ref$ ls -l

A5. Align the 1st pair of sequences to the reference genome using Burrows-Wheeler Aligner (BWA)

Check the fastq file contents

~/gatk_data_ref$ zcat ERR251295_1.fastq.gz | head

~/gatk_data_ref$ zcat ERR251295_2.fastq.gz | head -n 2

~/gatk-data-ref$ bwa mem -M -t 24 -R ‘@RG:ERR251295:Z:illumina:FCC1H7WACXX:HG02759’ Homo_sapiens_assembly38.fasta ERR251295_1.fastq.gz ERR251295_2.fastq.gz | samtools view -Sbh | samtools sort -o ERR251295_1.bam - && samtools index ERR251295_1.bam && samtools flagstat ERR251295_1.bam

Additional directory contents

~/gatk-data-ref$ ls -l

A6. Validate the BAM file and fix it if necessary

~/gatk-data-ref$ gatk ValidateSamFile -R Homo_sapiens_assembly38.fasta -I ERR251295_1.bam -M SUMMARY -O summary_ERR251295_1

Additional directory contents

~/gatk-data-ref$ cat summary_ERR251295_1

A7. Fix the error in the BAM file using samtools

~/gatk-data-ref$ samtools calmd -bAr ERR251295_1.bam Homo_sapiens_assembly38.fasta > ERR251295_1.calmd.bam

Additional directory contents

~/gatk-data-ref$ ls -l

A8. Mark duplicates

~/gatk-data-ref$ gatk MarkDuplicates -I ERR251295_1.calmd.bam -O ERR251295_1.calmd.mdup.bam -M ERR251295_1.calmd.dupMetrics.txt

Additional directory contents

~/gatk-data-ref$ ls -l

A9. Index the BAM file

~/gatk-data-ref$ samtools index ERR251295_1.calmd.mdup.bam

Additional directory contents

~/gatk-data-ref$ ls -l

A10. Run gatk HaplotypeCaller

~/gatk-data-ref$ gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR251295_1.calmd.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR251295_1.calmd.mdup.vcf

Additional directory contents

~/gatk-data-ref$ ls -l

A11. Record short variants (i.e., SNPs and indels) in the intended chromosomal region

~/gatk-data-ref$ bcftools stats ERR251295_1.calmd.mdup.vcf

B1. Align the 2nd pair of sequences to the reference genome using BWA and analyze as above

File contents

~/gatk-data-ref$ zcat ERR251296_1.fastq.gz | head -n 2

~/gatk-data-ref$ zcat ERR251296_2.fastq.gz | head -n 2

~/gatk-data-ref$ bwa mem -M -t 24 -R ‘@RG:ERR251296:Z:illumina:FCC1H7WACXX:HG02759’ Homo_sapiens_assembly38.fasta ERR251296_1.fastq.gz ERR251296_2.fastq.gz | samtools view -Sbh | samtools sort -o ERR251296_1.bam - && samtools index ERR251296_1.bam && samtools flagstat ERR251296_1.bam

B2. Validate BAM file and fix it if necessary

~/gatk-data-ref$ gatk ValidateSamFile -R Homo_sapiens_assembly38.fasta -I ERR251296_1.bam -M SUMMARY -O summary_ERR251296_1

~/gatk-data-ref$ cat summary_ERR251296_1

B3. Fix the error (INVALID_TAG_NM)

~/gatk-data-ref$ samtools calmd -bAr ERR251296_1.bam Homo_sapiens_assembly38.fasta > ERR251296_1.calmd.bam

B4. Mark duplicates

~/gatk-data-ref$ gatk MarkDuplicates -I ERR251296_1.calmd.bam -O ERR251296_1.calmd.mdup.bam -M ERR251296_1.calmd.dupMetrics.txt

B5. Index the BAM file

~/gatk-data-ref$ samtools index ERR251296_1.calmd.mdup.bam

B6. Run gatk HaplotypeCaller

~/gatk-data-ref$ gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR251296_1.calmd.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR251296_1.calmd.mdup.vcf

B7. Record short variants (SNPs and indels)

~/gatk-data-ref$ bcftools stats ERR251296_1.calmd.mdup.vcf

B8. Additional directory contents as a result of 2nd pair of sequence alignment

~/gatk-data-ref$ ls -l

BAM files could be merged and analyzed for combined analysis of multiple sequencing pairs of the same genome

C1. Merge the two BAM files

~/gatk-data-ref$ gatk MergeSamFiles -I ERR251295_1.calmd.bam -I ERR251296_1.calmd.bam -O ERR25129596.bam

C2. Mark duplicates

~/gatk-data-ref$ gatk MarkDuplicates -I ERR25129596.bam -O ERR25129596.mdup.bam -M ERR25129596.dupMetrics.txt

C3. Index the BAM file

samtools index ERR25129596.mdup.bam

C4. Run gatk HaplotypeCaller

gatk HaplotypeCaller -R Homo_sapiens_assembly38.fasta -I ERR25129596.mdup.bam -L chr20:15,800,000-16,100,000 -O ERR25129596.mdup.vcf