Answers.Body: <p>I am getting the following error using the BCFTOOLS COMMAND:
  $&gt;bcftools mpileup -Ob -o &lt;study.bcf&gt; -f &lt;ref.fa&gt; &lt;sample1.bam&gt;</p>
  <p>bcftools: bam2bcf.c:421: bcf_call_glfgen: Assertion <span class="math-container">`</span>epos&gt;=0 &amp;&amp; eposnpos' failed.</p>
  
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Answers.Text: As it’s currently written, your answer is unclear. Please [edit] to add additional details that will help others understand how this addresses the question asked. You can find more information on how to write good answers [in the help center](/help/how-to-answer).
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Answers.UserDisplayName: user22673
Answers.Body: <p>Recently, I also had a similar question and got my answer from Biostars. See my post and the answer I received: <a href="https://www.biostars.org/p/9508767/#9508880" rel="nofollow noreferrer">Germline variant calling from BAM without reference</a>.</p>
  <p>Basically, I was also wondering if it is possible to call SNVs/indels from <strong>aligned</strong> BAM files without reference because reference sequence was already used to produce such files. To this end, it appears some BAM files contain useful (but optional) read-level tags such as <code>MD</code> which, if present, would allow one to even reconstruct the entire reference sequence. For more details, please visit the link above.</p>
  
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Answers.CreationDate: 2022-02-16T23:42:45.510Z
Answers.LastActivityDate: 2022-02-16T23:42:45.510Z
Answers.UserDisplayName: Seung-been Steven Lee
Answers.Body: <p>To go from <code>.bam</code> to <code>.vcf</code>, it is fairly easy using <code>bcftools call</code>:</p>
  <pre><code>bcftools mpileup -Ob -o &lt;study.bcf&gt; -f &lt;ref.fa&gt; &lt;sample1.bam&gt; 
  bcftools call -vmO z -o &lt;study.vcf.gz&gt; &lt;study.bcf&gt;
  </code></pre>
  <p>Taken from <a href="http://www.htslib.org/workflow/wgs-call.html" rel="noreferrer">htslib website</a>.</p>
  
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Answers.CreationDate: 2022-02-23T17:39:49.713Z
Answers.Text: Does this method is applicable when I only have BAM files without bcf files?
Answers.UserDisplayName: Scott XU
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Answers.CreationDate: 2022-02-23T18:00:25.663Z
Answers.Text: @scott - the bcf (it’s the same as a vcf) is the output, not the input, so you’re fine to use it.
Answers.UserDisplayName: user438383
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Answers.CreationDate: 2022-02-23T21:31:55.237Z
Answers.Text: Hi~~ When I actually tried this method, I get some output like [E::faidx_adjust_position] The sequence "chr1_KI270706v1_random" was not found. Do you know how to fix it? Thanks a lot~~~
Answers.UserDisplayName: Scott XU
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Answers.UserDisplayName: user438383
Body: <p>I know that there is a lot of methods to do so, but most of them need to have a reference file. (For example, the GATK force me to provide a reference.)
  However, I think the BAM file I am working with already embedded the reference information in it. I tried to use the Samtool (mpileup) to do that, but it seems that the vcf file I get has different formats compared with the VCF documentations.
  Could anyone give me some advice?</p>
  <p>Also, could anyone provide a method to validate the vcf file without using a reference file?</p>
  <p>Thanks a lot!
  The following is what I got when running:</p>
  <pre><code>samtools view -H
  </code></pre>
  <pre><code>@HD VN:1.5  GO:none SO:coordinate
  @SQ SN:chr1 LN:248956422
  @SQ SN:chr2 LN:242193529
  @SQ SN:chr3 LN:198295559
  @SQ SN:chr4 LN:190214555
  @SQ SN:chr5 LN:181538259
  @SQ SN:chr6 LN:170805979
  @SQ SN:chr7 LN:159345973
  @SQ SN:chr8 LN:145138636
  @SQ SN:chr9 LN:138394717
  @SQ SN:chr10    LN:133797422
  @SQ SN:chr11    LN:135086622
  @SQ SN:chr12    LN:133275309
  @SQ SN:chr13    LN:114364328
  @SQ SN:chr14    LN:107043718
  @SQ SN:chr15    LN:101991189
  @SQ SN:chr16    LN:90338345
  @SQ SN:chr17    LN:83257441
  @SQ SN:chr18    LN:80373285
  @SQ SN:chr19    LN:58617616
  @SQ SN:chr20    LN:64444167
  @SQ SN:chr21    LN:46709983
  @SQ SN:chr22    LN:50818468
  @SQ SN:chrX LN:156040895
  @SQ SN:chrY LN:57227415
  @SQ SN:chrM LN:16569
  @SQ SN:chr1_KI270706v1_random   LN:175055
  @SQ SN:chr1_KI270707v1_random   LN:32032
  @SQ SN:chr1_KI270708v1_random   LN:127682
  @SQ SN:chr1_KI270709v1_random   LN:66860
  @SQ SN:chr1_KI270710v1_random   LN:40176
  @SQ SN:chr1_KI270711v1_random   LN:42210
  @SQ SN:chr1_KI270712v1_random   LN:176043
  @SQ SN:chr1_KI270713v1_random   LN:40745
  @SQ SN:chr1_KI270714v1_random   LN:41717
  @SQ SN:chr2_KI270715v1_random   LN:161471
  @SQ SN:chr2_KI270716v1_random   LN:153799
  @SQ SN:chr3_GL000221v1_random   LN:155397
  @SQ SN:chr4_GL000008v2_random   LN:209709
  @SQ SN:chr5_GL000208v1_random   LN:92689
  @SQ SN:chr9_KI270717v1_random   LN:40062
  @SQ SN:chr9_KI270718v1_random   LN:38054
  @SQ SN:chr9_KI270719v1_random   LN:176845
  @SQ SN:chr9_KI270720v1_random   LN:39050
  @SQ SN:chr11_KI270721v1_random  LN:100316
  @SQ SN:chr14_GL000009v2_random  LN:201709
  @SQ SN:chr14_GL000225v1_random  LN:211173
  @SQ SN:chr14_KI270722v1_random  LN:194050
  @SQ SN:chr14_GL000194v1_random  LN:191469
  @SQ SN:chr14_KI270723v1_random  LN:38115
  @SQ SN:chr14_KI270724v1_random  LN:39555
  @SQ SN:chr14_KI270725v1_random  LN:172810
  @SQ SN:chr14_KI270726v1_random  LN:43739
  @SQ SN:chr15_KI270727v1_random  LN:448248
  @SQ SN:chr16_KI270728v1_random  LN:1872759
  @SQ SN:chr17_GL000205v2_random  LN:185591
  @SQ SN:chr17_KI270729v1_random  LN:280839
  @SQ SN:chr17_KI270730v1_random  LN:112551
  @SQ SN:chr22_KI270731v1_random  LN:150754
  @SQ SN:chr22_KI270732v1_random  LN:41543
  @SQ SN:chr22_KI270733v1_random  LN:179772
  @SQ SN:chr22_KI270734v1_random  LN:165050
  @SQ SN:chr22_KI270735v1_random  LN:42811
  @SQ SN:chr22_KI270736v1_random  LN:181920
  @SQ SN:chr22_KI270737v1_random  LN:103838
  @SQ SN:chr22_KI270738v1_random  LN:99375
  @SQ SN:chr22_KI270739v1_random  LN:73985
  @SQ SN:chrY_KI270740v1_random   LN:37240
  @SQ SN:chrUn_KI270302v1 LN:2274
  @SQ SN:chrUn_KI270304v1 LN:2165
  @SQ SN:chrUn_KI270303v1 LN:1942
  @SQ SN:chrUn_KI270305v1 LN:1472
  @SQ SN:chrUn_KI270322v1 LN:21476
  @SQ SN:chrUn_KI270320v1 LN:4416
  @SQ SN:chrUn_KI270310v1 LN:1201
  @SQ SN:chrUn_KI270316v1 LN:1444
  @SQ SN:chrUn_KI270315v1 LN:2276
  @SQ SN:chrUn_KI270312v1 LN:998
  @SQ SN:chrUn_KI270311v1 LN:12399
  @SQ SN:chrUn_KI270317v1 LN:37690
  @SQ SN:chrUn_KI270412v1 LN:1179
  @SQ SN:chrUn_KI270411v1 LN:2646
  @SQ SN:chrUn_KI270414v1 LN:2489
  @SQ SN:chrUn_KI270419v1 LN:1029
  @SQ SN:chrUn_KI270418v1 LN:2145
  @SQ SN:chrUn_KI270420v1 LN:2321
  @SQ SN:chrUn_KI270424v1 LN:2140
  @SQ SN:chrUn_KI270417v1 LN:2043
  @SQ SN:chrUn_KI270422v1 LN:1445
  @SQ SN:chrUn_KI270423v1 LN:981
  @SQ SN:chrUn_KI270425v1 LN:1884
  @SQ SN:chrUn_KI270429v1 LN:1361
  @SQ SN:chrUn_KI270442v1 LN:392061
  @SQ SN:chrUn_KI270466v1 LN:1233
  @SQ SN:chrUn_KI270465v1 LN:1774
  @SQ SN:chrUn_KI270467v1 LN:3920
  @SQ SN:chrUn_KI270435v1 LN:92983
  @SQ SN:chrUn_KI270438v1 LN:112505
  @SQ SN:chrUn_KI270468v1 LN:4055
  @SQ SN:chrUn_KI270510v1 LN:2415
  @SQ SN:chrUn_KI270509v1 LN:2318
  @SQ SN:chrUn_KI270518v1 LN:2186
  @SQ SN:chrUn_KI270508v1 LN:1951
  @SQ SN:chrUn_KI270516v1 LN:1300
  @SQ SN:chrUn_KI270512v1 LN:22689
  @SQ SN:chrUn_KI270519v1 LN:138126
  @SQ SN:chrUn_KI270522v1 LN:5674
  @SQ SN:chrUn_KI270511v1 LN:8127
  @SQ SN:chrUn_KI270515v1 LN:6361
  @SQ SN:chrUn_KI270507v1 LN:5353
  @SQ SN:chrUn_KI270517v1 LN:3253
  @SQ SN:chrUn_KI270529v1 LN:1899
  @SQ SN:chrUn_KI270528v1 LN:2983
  @SQ SN:chrUn_KI270530v1 LN:2168
  @SQ SN:chrUn_KI270539v1 LN:993
  @SQ SN:chrUn_KI270538v1 LN:91309
  @SQ SN:chrUn_KI270544v1 LN:1202
  @SQ SN:chrUn_KI270548v1 LN:1599
  @SQ SN:chrUn_KI270583v1 LN:1400
  @SQ SN:chrUn_KI270587v1 LN:2969
  @SQ SN:chrUn_KI270580v1 LN:1553
  @SQ SN:chrUn_KI270581v1 LN:7046
  @SQ SN:chrUn_KI270579v1 LN:31033
  @SQ SN:chrUn_KI270589v1 LN:44474
  @SQ SN:chrUn_KI270590v1 LN:4685
  @SQ SN:chrUn_KI270584v1 LN:4513
  @SQ SN:chrUn_KI270582v1 LN:6504
  @SQ SN:chrUn_KI270588v1 LN:6158
  @SQ SN:chrUn_KI270593v1 LN:3041
  @SQ SN:chrUn_KI270591v1 LN:5796
  @SQ SN:chrUn_KI270330v1 LN:1652
  @SQ SN:chrUn_KI270329v1 LN:1040
  @SQ SN:chrUn_KI270334v1 LN:1368
  @SQ SN:chrUn_KI270333v1 LN:2699
  @SQ SN:chrUn_KI270335v1 LN:1048
  @SQ SN:chrUn_KI270338v1 LN:1428
  @SQ SN:chrUn_KI270340v1 LN:1428
  @SQ SN:chrUn_KI270336v1 LN:1026
  @SQ SN:chrUn_KI270337v1 LN:1121
  @SQ SN:chrUn_KI270363v1 LN:1803
  @SQ SN:chrUn_KI270364v1 LN:2855
  @SQ SN:chrUn_KI270362v1 LN:3530
  @SQ SN:chrUn_KI270366v1 LN:8320
  @SQ SN:chrUn_KI270378v1 LN:1048
  @SQ SN:chrUn_KI270379v1 LN:1045
  @SQ SN:chrUn_KI270389v1 LN:1298
  @SQ SN:chrUn_KI270390v1 LN:2387
  @SQ SN:chrUn_KI270387v1 LN:1537
  @SQ SN:chrUn_KI270395v1 LN:1143
  @SQ SN:chrUn_KI270396v1 LN:1880
  @SQ SN:chrUn_KI270388v1 LN:1216
  @SQ SN:chrUn_KI270394v1 LN:970
  @SQ SN:chrUn_KI270386v1 LN:1788
  @SQ SN:chrUn_KI270391v1 LN:1484
  @SQ SN:chrUn_KI270383v1 LN:1750
  @SQ SN:chrUn_KI270393v1 LN:1308
  @SQ SN:chrUn_KI270384v1 LN:1658
  @SQ SN:chrUn_KI270392v1 LN:971
  @SQ SN:chrUn_KI270381v1 LN:1930
  @SQ SN:chrUn_KI270385v1 LN:990
  @SQ SN:chrUn_KI270382v1 LN:4215
  @SQ SN:chrUn_KI270376v1 LN:1136
  @SQ SN:chrUn_KI270374v1 LN:2656
  @SQ SN:chrUn_KI270372v1 LN:1650
  @SQ SN:chrUn_KI270373v1 LN:1451
  @SQ SN:chrUn_KI270375v1 LN:2378
  @SQ SN:chrUn_KI270371v1 LN:2805
  @SQ SN:chrUn_KI270448v1 LN:7992
  @SQ SN:chrUn_KI270521v1 LN:7642
  @SQ SN:chrUn_GL000195v1 LN:182896
  @SQ SN:chrUn_GL000219v1 LN:179198
  @SQ SN:chrUn_GL000220v1 LN:161802
  @SQ SN:chrUn_GL000224v1 LN:179693
  @SQ SN:chrUn_KI270741v1 LN:157432
  @SQ SN:chrUn_GL000226v1 LN:15008
  @SQ SN:chrUn_GL000213v1 LN:164239
  @SQ SN:chrUn_KI270743v1 LN:210658
  @SQ SN:chrUn_KI270744v1 LN:168472
  @SQ SN:chrUn_KI270745v1 LN:41891
  @SQ SN:chrUn_KI270746v1 LN:66486
  @SQ SN:chrUn_KI270747v1 LN:198735
  @SQ SN:chrUn_KI270748v1 LN:93321
  @SQ SN:chrUn_KI270749v1 LN:158759
  @SQ SN:chrUn_KI270750v1 LN:148850
  @SQ SN:chrUn_KI270751v1 LN:150742
  @SQ SN:chrUn_KI270752v1 LN:27745
  @SQ SN:chrUn_KI270753v1 LN:62944
  @SQ SN:chrUn_KI270754v1 LN:40191
  @SQ SN:chrUn_KI270755v1 LN:36723
  @SQ SN:chrUn_KI270756v1 LN:79590
  @SQ SN:chrUn_KI270757v1 LN:71251
  @SQ SN:chrUn_GL000214v1 LN:137718
  @SQ SN:chrUn_KI270742v1 LN:186739
  @SQ SN:chrUn_GL000216v2 LN:176608
  @SQ SN:chrUn_GL000218v1 LN:161147
  @SQ SN:chrEBV   LN:171823
  @RG ID:Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1  CN:Novogene LB:Wit_Exon9_DEL    PL:illuminPU:Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1    SM:Wit_Exon9_DEL
  @PG ID:bwa  PN:bwa  CL:bwa mem -t 8 -M -R @RG\tID:Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1\tSM:Wit_Exon9_DEL\tLB:Wit_Exon9_DEL\tPU:Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1\tPL:illumina\tCN:Novogene /PUBLIC/source/HW/Disease/database/genome/human/hg38/bwa_index_whole/hg38.fa /TJPROJ6/GB_HUMAN/PJ_AI/cancer/auto-WES-Somatic/1000/X202SC2005/X202SC20050050-Z01/F002/QC/Wit_Exon9_DEL/Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1_1.clean.fq.gz /TJPROJ6/GB_HUMAN/PJ_AI/cancer/auto-WES-Somatic/1000/X202SC2005/X202SC20050050-Z01/F002/QC/Wit_Exon9_DEL/Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1_2.clean.fq.gz  VN:0.7.17-r1188
  @PG ID:sambamba CL:view -S -f bam /dev/stdin    PP:bwa  VN:1.0
  @PG ID:MarkDuplicates   VN:2.18.9-SNAPSHOT  CL:MarkDuplicates INPUT=[/TJPROJ6/GB_HUMAN/PJ_AI/service/cromwell/cromwell-executions/ToSomatic/fddd223f-25fd-4533-ae86-14e106c58ec5/call-Mapping/shard-0/Mapping/14f30bea-add3-4ead-b7e1-fcb202ae18f5/call-picardMarkdup/inputs/-714370009/Wit_Exon9_DEL.merge.bam] OUTPUT=/TJPROJ6/GB_HUMAN/PJ_AI/cancer/auto-WES-Somatic/1000/X202SC2005/X202SC20050050-Z01/F002/Mapping/Wit_Exon9_DEL/Wit_Exon9_DEL.final.bam METRICS_FILE=metric.txt REMOVE_DUPLICATES=false ASSUME_SORTED=true OPTICAL_DUPLICATE_PIXEL_DISTANCE=2500 TMP_DIR=[.] VALIDATION_STRINGENCY=SILENT MAX_RECORDS_IN_RAM=4000000 CREATE_INDEX=true    MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP=50000 MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=8000 SORTING_COLLECTION_SIZE_RATIO=0.25 TAG_DUPLICATE_SET_MEMBERS=false REMOVE_SEQUENCING_DUPLICATES=false TAGGING_POLICY=DontTag CLEAR_DT=true ADD_PG_TAG_TO_READS=true DUPLICATE_SCORING_STRATEGY=SUM_OF_BASE_QUALITIES PROGRAM_RECORD_ID=MarkDuplicates PROGRAM_GROUP_NAME=MarkDuplicates READ_NAME_REGEX=&lt;optimized capture of last three ':' separated fields as numeric values&gt; MAX_OPTICAL_DUPLICATE_SET_SIZE=300000 VERBOSITY=INFO QUIET=false COMPRESSION_LEVEL=5 CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json USE_JDK_DEFLATER=false USE_JDK_INFLATER=false   PN:MarkDuplicates
  @PG ID:samtools PN:samtools PP:sambamba VN:1.10 CL:samtools view -H /Users/XXXX/Desktop/data/02.Bam/XXXX.final.bam
  @PG ID:samtools.1   PN:samtools PP:MarkDuplicates   VN:1.10 CL:samtools view -H /Users/XXXX/Desktop/data/02.Bam/XXXX.final.bam
  </code></pre>
  <p>The output file I get when using Samtools looks like: <a href="https://i.sstatic.net/8dIfA.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/8dIfA.png" alt="enter image description here" /></a></p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-01-11T23:56:38.550Z
Comments.Text: This is generally a bad idea....if you have a reference, use it! otherwise your VCF will be wrong, for example, you'll miss all the HOM_VAR variants.
Comments.UserDisplayName: Yossi Farjoun
Tags: <sam><vcf><gatk>
Title: How to convert a BAM file into a VCF file
ThreadText: # How to convert a BAM file into a VCF file
  
  (Asked by: username_0 on 2022-02-16)
  
  I know that there is a lot of methods to do so, but most of them need to have a reference file. (For example, the GATK force me to provide a reference.)
  However, I think the BAM file I am working with already embedded the reference information in it. I tried to use the Samtool (mpileup) to do that, but it seems that the vcf file I get has different formats compared with the VCF documentations.
  Could anyone give me some advice?
  
  Also, could anyone provide a method to validate the vcf file without using a reference file?
  
  Thanks a lot!
  The following is what I got when running:
  
  ```
  samtools view -H
  
  ```
  
  ```
  @HD VN:1.5  GO:none SO:coordinate
  @SQ SN:chr1 LN:248956422
  @SQ SN:chr2 LN:242193529
  @SQ SN:chr3 LN:198295559
  @SQ SN:chr4 LN:190214555
  @SQ SN:chr5 LN:181538259
  @SQ SN:chr6 LN:170805979
  @SQ SN:chr7 LN:159345973
  @SQ SN:chr8 LN:145138636
  @SQ SN:chr9 LN:138394717
  @SQ SN:chr10    LN:133797422
  @SQ SN:chr11    LN:135086622
  @SQ SN:chr12    LN:133275309
  @SQ SN:chr13    LN:114364328
  @SQ SN:chr14    LN:107043718
  @SQ SN:chr15    LN:101991189
  @SQ SN:chr16    LN:90338345
  @SQ SN:chr17    LN:83257441
  @SQ SN:chr18    LN:80373285
  @SQ SN:chr19    LN:58617616
  @SQ SN:chr20    LN:64444167
  @SQ SN:chr21    LN:46709983
  @SQ SN:chr22    LN:50818468
  @SQ SN:chrX LN:156040895
  @SQ SN:chrY LN:57227415
  @SQ SN:chrM LN:16569
  @SQ SN:chr1_KI270706v1_random   LN:175055
  @SQ SN:chr1_KI270707v1_random   LN:32032
  @SQ SN:chr1_KI270708v1_random   LN:127682
  @SQ SN:chr1_KI270709v1_random   LN:66860
  @SQ SN:chr1_KI270710v1_random   LN:40176
  @SQ SN:chr1_KI270711v1_random   LN:42210
  @SQ SN:chr1_KI270712v1_random   LN:176043
  @SQ SN:chr1_KI270713v1_random   LN:40745
  @SQ SN:chr1_KI270714v1_random   LN:41717
  @SQ SN:chr2_KI270715v1_random   LN:161471
  @SQ SN:chr2_KI270716v1_random   LN:153799
  @SQ SN:chr3_GL000221v1_random   LN:155397
  @SQ SN:chr4_GL000008v2_random   LN:209709
  @SQ SN:chr5_GL000208v1_random   LN:92689
  @SQ SN:chr9_KI270717v1_random   LN:40062
  @SQ SN:chr9_KI270718v1_random   LN:38054
  @SQ SN:chr9_KI270719v1_random   LN:176845
  @SQ SN:chr9_KI270720v1_random   LN:39050
  @SQ SN:chr11_KI270721v1_random  LN:100316
  @SQ SN:chr14_GL000009v2_random  LN:201709
  @SQ SN:chr14_GL000225v1_random  LN:211173
  @SQ SN:chr14_KI270722v1_random  LN:194050
  @SQ SN:chr14_GL000194v1_random  LN:191469
  @SQ SN:chr14_KI270723v1_random  LN:38115
  @SQ SN:chr14_KI270724v1_random  LN:39555
  @SQ SN:chr14_KI270725v1_random  LN:172810
  @SQ SN:chr14_KI270726v1_random  LN:43739
  @SQ SN:chr15_KI270727v1_random  LN:448248
  @SQ SN:chr16_KI270728v1_random  LN:1872759
  @SQ SN:chr17_GL000205v2_random  LN:185591
  @SQ SN:chr17_KI270729v1_random  LN:280839
  @SQ SN:chr17_KI270730v1_random  LN:112551
  @SQ SN:chr22_KI270731v1_random  LN:150754
  @SQ SN:chr22_KI270732v1_random  LN:41543
  @SQ SN:chr22_KI270733v1_random  LN:179772
  @SQ SN:chr22_KI270734v1_random  LN:165050
  @SQ SN:chr22_KI270735v1_random  LN:42811
  @SQ SN:chr22_KI270736v1_random  LN:181920
  @SQ SN:chr22_KI270737v1_random  LN:103838
  @SQ SN:chr22_KI270738v1_random  LN:99375
  @SQ SN:chr22_KI270739v1_random  LN:73985
  @SQ SN:chrY_KI270740v1_random   LN:37240
  @SQ SN:chrUn_KI270302v1 LN:2274
  @SQ SN:chrUn_KI270304v1 LN:2165
  @SQ SN:chrUn_KI270303v1 LN:1942
  @SQ SN:chrUn_KI270305v1 LN:1472
  @SQ SN:chrUn_KI270322v1 LN:21476
  @SQ SN:chrUn_KI270320v1 LN:4416
  @SQ SN:chrUn_KI270310v1 LN:1201
  @SQ SN:chrUn_KI270316v1 LN:1444
  @SQ SN:chrUn_KI270315v1 LN:2276
  @SQ SN:chrUn_KI270312v1 LN:998
  @SQ SN:chrUn_KI270311v1 LN:12399
  @SQ SN:chrUn_KI270317v1 LN:37690
  @SQ SN:chrUn_KI270412v1 LN:1179
  @SQ SN:chrUn_KI270411v1 LN:2646
  @SQ SN:chrUn_KI270414v1 LN:2489
  @SQ SN:chrUn_KI270419v1 LN:1029
  @SQ SN:chrUn_KI270418v1 LN:2145
  @SQ SN:chrUn_KI270420v1 LN:2321
  @SQ SN:chrUn_KI270424v1 LN:2140
  @SQ SN:chrUn_KI270417v1 LN:2043
  @SQ SN:chrUn_KI270422v1 LN:1445
  @SQ SN:chrUn_KI270423v1 LN:981
  @SQ SN:chrUn_KI270425v1 LN:1884
  @SQ SN:chrUn_KI270429v1 LN:1361
  @SQ SN:chrUn_KI270442v1 LN:392061
  @SQ SN:chrUn_KI270466v1 LN:1233
  @SQ SN:chrUn_KI270465v1 LN:1774
  @SQ SN:chrUn_KI270467v1 LN:3920
  @SQ SN:chrUn_KI270435v1 LN:92983
  @SQ SN:chrUn_KI270438v1 LN:112505
  @SQ SN:chrUn_KI270468v1 LN:4055
  @SQ SN:chrUn_KI270510v1 LN:2415
  @SQ SN:chrUn_KI270509v1 LN:2318
  @SQ SN:chrUn_KI270518v1 LN:2186
  @SQ SN:chrUn_KI270508v1 LN:1951
  @SQ SN:chrUn_KI270516v1 LN:1300
  @SQ SN:chrUn_KI270512v1 LN:22689
  @SQ SN:chrUn_KI270519v1 LN:138126
  @SQ SN:chrUn_KI270522v1 LN:5674
  @SQ SN:chrUn_KI270511v1 LN:8127
  @SQ SN:chrUn_KI270515v1 LN:6361
  @SQ SN:chrUn_KI270507v1 LN:5353
  @SQ SN:chrUn_KI270517v1 LN:3253
  @SQ SN:chrUn_KI270529v1 LN:1899
  @SQ SN:chrUn_KI270528v1 LN:2983
  @SQ SN:chrUn_KI270530v1 LN:2168
  @SQ SN:chrUn_KI270539v1 LN:993
  @SQ SN:chrUn_KI270538v1 LN:91309
  @SQ SN:chrUn_KI270544v1 LN:1202
  @SQ SN:chrUn_KI270548v1 LN:1599
  @SQ SN:chrUn_KI270583v1 LN:1400
  @SQ SN:chrUn_KI270587v1 LN:2969
  @SQ SN:chrUn_KI270580v1 LN:1553
  @SQ SN:chrUn_KI270581v1 LN:7046
  @SQ SN:chrUn_KI270579v1 LN:31033
  @SQ SN:chrUn_KI270589v1 LN:44474
  @SQ SN:chrUn_KI270590v1 LN:4685
  @SQ SN:chrUn_KI270584v1 LN:4513
  @SQ SN:chrUn_KI270582v1 LN:6504
  @SQ SN:chrUn_KI270588v1 LN:6158
  @SQ SN:chrUn_KI270593v1 LN:3041
  @SQ SN:chrUn_KI270591v1 LN:5796
  @SQ SN:chrUn_KI270330v1 LN:1652
  @SQ SN:chrUn_KI270329v1 LN:1040
  @SQ SN:chrUn_KI270334v1 LN:1368
  @SQ SN:chrUn_KI270333v1 LN:2699
  @SQ SN:chrUn_KI270335v1 LN:1048
  @SQ SN:chrUn_KI270338v1 LN:1428
  @SQ SN:chrUn_KI270340v1 LN:1428
  @SQ SN:chrUn_KI270336v1 LN:1026
  @SQ SN:chrUn_KI270337v1 LN:1121
  @SQ SN:chrUn_KI270363v1 LN:1803
  @SQ SN:chrUn_KI270364v1 LN:2855
  @SQ SN:chrUn_KI270362v1 LN:3530
  @SQ SN:chrUn_KI270366v1 LN:8320
  @SQ SN:chrUn_KI270378v1 LN:1048
  @SQ SN:chrUn_KI270379v1 LN:1045
  @SQ SN:chrUn_KI270389v1 LN:1298
  @SQ SN:chrUn_KI270390v1 LN:2387
  @SQ SN:chrUn_KI270387v1 LN:1537
  @SQ SN:chrUn_KI270395v1 LN:1143
  @SQ SN:chrUn_KI270396v1 LN:1880
  @SQ SN:chrUn_KI270388v1 LN:1216
  @SQ SN:chrUn_KI270394v1 LN:970
  @SQ SN:chrUn_KI270386v1 LN:1788
  @SQ SN:chrUn_KI270391v1 LN:1484
  @SQ SN:chrUn_KI270383v1 LN:1750
  @SQ SN:chrUn_KI270393v1 LN:1308
  @SQ SN:chrUn_KI270384v1 LN:1658
  @SQ SN:chrUn_KI270392v1 LN:971
  @SQ SN:chrUn_KI270381v1 LN:1930
  @SQ SN:chrUn_KI270385v1 LN:990
  @SQ SN:chrUn_KI270382v1 LN:4215
  @SQ SN:chrUn_KI270376v1 LN:1136
  @SQ SN:chrUn_KI270374v1 LN:2656
  @SQ SN:chrUn_KI270372v1 LN:1650
  @SQ SN:chrUn_KI270373v1 LN:1451
  @SQ SN:chrUn_KI270375v1 LN:2378
  @SQ SN:chrUn_KI270371v1 LN:2805
  @SQ SN:chrUn_KI270448v1 LN:7992
  @SQ SN:chrUn_KI270521v1 LN:7642
  @SQ SN:chrUn_GL000195v1 LN:182896
  @SQ SN:chrUn_GL000219v1 LN:179198
  @SQ SN:chrUn_GL000220v1 LN:161802
  @SQ SN:chrUn_GL000224v1 LN:179693
  @SQ SN:chrUn_KI270741v1 LN:157432
  @SQ SN:chrUn_GL000226v1 LN:15008
  @SQ SN:chrUn_GL000213v1 LN:164239
  @SQ SN:chrUn_KI270743v1 LN:210658
  @SQ SN:chrUn_KI270744v1 LN:168472
  @SQ SN:chrUn_KI270745v1 LN:41891
  @SQ SN:chrUn_KI270746v1 LN:66486
  @SQ SN:chrUn_KI270747v1 LN:198735
  @SQ SN:chrUn_KI270748v1 LN:93321
  @SQ SN:chrUn_KI270749v1 LN:158759
  @SQ SN:chrUn_KI270750v1 LN:148850
  @SQ SN:chrUn_KI270751v1 LN:150742
  @SQ SN:chrUn_KI270752v1 LN:27745
  @SQ SN:chrUn_KI270753v1 LN:62944
  @SQ SN:chrUn_KI270754v1 LN:40191
  @SQ SN:chrUn_KI270755v1 LN:36723
  @SQ SN:chrUn_KI270756v1 LN:79590
  @SQ SN:chrUn_KI270757v1 LN:71251
  @SQ SN:chrUn_GL000214v1 LN:137718
  @SQ SN:chrUn_KI270742v1 LN:186739
  @SQ SN:chrUn_GL000216v2 LN:176608
  @SQ SN:chrUn_GL000218v1 LN:161147
  @SQ SN:chrEBV   LN:171823
  @RG ID:Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1  CN:Novogene LB:Wit_Exon9_DEL    PL:illuminPU:Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1    SM:Wit_Exon9_DEL
  @PG ID:bwa  PN:bwa  CL:bwa mem -t 8 -M -R @RG\tID:Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1\tSM:Wit_Exon9_DEL\tLB:Wit_Exon9_DEL\tPU:Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1\tPL:illumina\tCN:Novogene /PUBLIC/source/HW/Disease/database/genome/human/hg38/bwa_index_whole/hg38.fa /TJPROJ6/GB_HUMAN/PJ_AI/cancer/auto-WES-Somatic/1000/X202SC2005/X202SC20050050-Z01/F002/QC/Wit_Exon9_DEL/Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1_1.clean.fq.gz /TJPROJ6/GB_HUMAN/PJ_AI/cancer/auto-WES-Somatic/1000/X202SC2005/X202SC20050050-Z01/F002/QC/Wit_Exon9_DEL/Wit_Exon9_DEL_FKDN202385493-1A_H5HHJDSXY_L1_2.clean.fq.gz  VN:0.7.17-r1188
  @PG ID:sambamba CL:view -S -f bam /dev/stdin    PP:bwa  VN:1.0
  @PG ID:MarkDuplicates   VN:2.18.9-SNAPSHOT  CL:MarkDuplicates INPUT=[/TJPROJ6/GB_HUMAN/PJ_AI/service/cromwell/cromwell-executions/ToSomatic/fddd223f-25fd-4533-ae86-14e106c58ec5/call-Mapping/shard-0/Mapping/14f30bea-add3-4ead-b7e1-fcb202ae18f5/call-picardMarkdup/inputs/-714370009/Wit_Exon9_DEL.merge.bam] OUTPUT=/TJPROJ6/GB_HUMAN/PJ_AI/cancer/auto-WES-Somatic/1000/X202SC2005/X202SC20050050-Z01/F002/Mapping/Wit_Exon9_DEL/Wit_Exon9_DEL.final.bam METRICS_FILE=metric.txt REMOVE_DUPLICATES=false ASSUME_SORTED=true OPTICAL_DUPLICATE_PIXEL_DISTANCE=2500 TMP_DIR=[.] VALIDATION_STRINGENCY=SILENT MAX_RECORDS_IN_RAM=4000000 CREATE_INDEX=true    MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP=50000 MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=8000 SORTING_COLLECTION_SIZE_RATIO=0.25 TAG_DUPLICATE_SET_MEMBERS=false REMOVE_SEQUENCING_DUPLICATES=false TAGGING_POLICY=DontTag CLEAR_DT=true ADD_PG_TAG_TO_READS=true DUPLICATE_SCORING_STRATEGY=SUM_OF_BASE_QUALITIES PROGRAM_RECORD_ID=MarkDuplicates PROGRAM_GROUP_NAME=MarkDuplicates READ_NAME_REGEX=<optimized capture of last three ':' separated fields as numeric values> MAX_OPTICAL_DUPLICATE_SET_SIZE=300000 VERBOSITY=INFO QUIET=false COMPRESSION_LEVEL=5 CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json USE_JDK_DEFLATER=false USE_JDK_INFLATER=false   PN:MarkDuplicates
  @PG ID:samtools PN:samtools PP:sambamba VN:1.10 CL:samtools view -H /Users/XXXX/Desktop/data/02.Bam/XXXX.final.bam
  @PG ID:samtools.1   PN:samtools PP:MarkDuplicates   VN:1.10 CL:samtools view -H /Users/XXXX/Desktop/data/02.Bam/XXXX.final.bam
  
  ```
  
  The output file I get when using Samtools looks like: [![enter image description here](https://i.sstatic.net/8dIfA.png)](https://i.sstatic.net/8dIfA.png)
  
  **Tags:** sam, vcf, gatk
  
  > **Comments:**
  > **username_1**: This is generally a bad idea....if you have a reference, use it! otherwise your VCF will be wrong, for example, you'll miss all the HOM_VAR variants.  
  
  ---
  
  ## 3 answers
  
  ### Answer by username_5 on 2022-02-17. Score: 5 | Accepted answer
  
  To go from `.bam` to `.vcf`, it is fairly easy using `bcftools call`:
  
  ```
  bcftools mpileup -Ob -o <study.bcf> -f <ref.fa> <sample1.bam> 
  bcftools call -vmO z -o <study.vcf.gz> <study.bcf>
  
  ```
  
  Taken from [htslib website](http://www.htslib.org/workflow/wgs-call.html).
  
  > **Comments:**
  > **username_0**: Does this method is applicable when I only have BAM files without bcf files?  
  > **username_5**: @username_0 - the bcf (it’s the same as a vcf) is the output, not the input, so you’re fine to use it.  
  > **username_0**: Hi~~ When I actually tried this method, I get some output like [E::faidx_adjust_position] The sequence "chr1_KI270706v1_random" was not found. Do you know how to fix it? Thanks a lot~~~  
  
  ---
  
  ### Answer by username_2 on 2024-12-24. Score: 0
  
  I am getting the following error using the BCFTOOLS COMMAND:
  $>bcftools mpileup -Ob -o <study.bcf> -f <ref.fa> <sample1.bam>
  
  bcftools: bam2bcf.c:421: bcf\_call\_glfgen: Assertion `epos>=0 && eposnpos' failed.
  
  > **Comments:**
  > **username_3**: As it’s currently written, your answer is unclear. Please [edit] to add additional details that will help others understand how this addresses the question asked. You can find more information on how to write good answers [in the help center](/help/how-to-answer).  
  
  ---
  
  ### Answer by username_4 on 2022-02-16. Score: -1
  
  Recently, I also had a similar question and got my answer from Biostars. See my post and the answer I received: [Germline variant calling from BAM without reference](https://www.biostars.org/p/9508767/#9508880).
  
  Basically, I was also wondering if it is possible to call SNVs/indels from **aligned** BAM files without reference because reference sequence was already used to produce such files. To this end, it appears some BAM files contain useful (but optional) read-level tags such as `MD` which, if present, would allow one to even reconstruct the entire reference sequence. For more details, please visit the link above.
  
  
  ---
  
  