text: user2869: Currently I'm performing whole analysis (pipeline from *.fastq to *.vcf) of 41  samples (targeted NGS). I rely on GATK best practices, however with some modifications. I decided to use the following tools:
  #mapping
  bwa mem (with mem - alternate haplotypes)
  
  #preprocessing
  RevertSam (Picard)
  MergeBamAlignment (Picard)
  MarkDuplicates (Picard)
  BaseRecalibrator (GATK)
  ApplyBQSR (GATK)
  AnalyzeCovariates (GATK)
  SortSam (Picard)
  
  #call, filter and annotate variants
  HaplotypeCaller (GATK)
  GenotypeGVCFs (GATK)
  snpEff, SnpSift
  
  Nevertheless, after marking the duplicates with MarkDuplicates about 99% of reads have been marked as duplicated, hence the coverage decreased from several hundreds to literally up to five. So I decided to omit this step.
  Have you ever encountered a similar problem? What is your opinion about this?
  Best Regards,
  Adam
  EDIT
  The Agilent Haloplex custom target enrichment kit (HaloPlex Target Enrichment for Illumina Kit) has been used to prepare the libraries, sequencing done using Illumina HiSeq 2500. Targeted NGS of all exons (only) of 90 genes.
  Here I provide how the final bam look like. This is the result without duplicate marking, otherwise there're only several reads per exon...
  
  
  user73: Most duplicate marking programs do it by looking at the start and end location of hits. In this case, if two reads have identical start and end locations for both reads, then they will be considered [PCR] duplicates of each other. When running through a duplicate filter, you will only get one read for each unique pair.
  This is much more likely for targeted sequencing, because the length covered by the template is much smaller. It's also more likely that you actually do have lots of PCR duplicates for the same reason.
  
  user2085: If you're doing target capture, especially of a small region, it is entirely possible that there are only five unique reads. Target capture is molecularly finicky and sometimes the entire region is not captured.
  
original_text: 2869: <p>Currently I'm performing whole analysis (pipeline from *.fastq to *.vcf) of 41  samples (targeted NGS). I rely on GATK best practices, however with some modifications. I decided to use the following tools:</p>
  <pre><code>#mapping
  bwa mem (with mem - alternate haplotypes)
  
  #preprocessing
  RevertSam (Picard)
  MergeBamAlignment (Picard)
  MarkDuplicates (Picard)
  BaseRecalibrator (GATK)
  ApplyBQSR (GATK)
  AnalyzeCovariates (GATK)
  SortSam (Picard)
  
  #call, filter and annotate variants
  HaplotypeCaller (GATK)
  GenotypeGVCFs (GATK)
  snpEff, SnpSift
  </code></pre>
  <p>Nevertheless, after marking the duplicates with <code>MarkDuplicates</code> about 99% of reads have been marked as duplicated, hence the coverage decreased from several hundreds to literally up to five. So I decided to omit this step.</p>
  <p>Have you ever encountered a similar problem? What is your opinion about this?</p>
  <p>Best Regards,
  Adam</p>
  <h1>EDIT</h1>
  <p>The Agilent Haloplex custom target enrichment kit (HaloPlex Target Enrichment for Illumina Kit) has been used to prepare the libraries, sequencing done using Illumina HiSeq 2500. Targeted NGS of all exons (only) of 90 genes.</p>
  <p>Here I provide how the final bam look like. This is the result without duplicate marking, otherwise there're only several reads per exon...
  <a href="https://i.stack.imgur.com/k8yze.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/k8yze.png" alt="enter image description here" /></a></p>
   73: <p>Most duplicate marking programs do it by looking at the start and end location of hits. In this case, if two reads have identical start and end locations for both reads, then they will be considered [PCR] duplicates of each other. When running through a duplicate filter, you will only get one read for each unique pair.</p>
  
  <p>This is much more likely for targeted sequencing, because the length covered by the template is much smaller. It's also more likely that you actually <em>do</em> have lots of PCR duplicates for the same reason.</p>
   2085: <p>If you're doing target capture, especially of a small region, it is entirely possible that there are only five unique reads. Target capture is molecularly finicky and sometimes the entire region is not captured.</p>
  