Samtools flagstat secondary

Samtools flagstat secondary. This table summarizes the command-line arguments that are specific to this tool. Let’s take a look at the first few lines of the original file. To consider also secondary alignments, BEDtools could be an alternative. 40% primary. The samtools flagstat tool provides counts for each of 13 read categories based primarily on bit flags in the FLAG field ( note that this refers to the SAMtools released version 1. 0x4 bit not set. 0x4, 0x100 and 0x800 bits not set paired in sequencing. fasta samtools fastq input. Mar 12, 2013 · Hi i m using tophat-cufflink pipeline for RNA seq data analysis. sam | in. Samtools和Bcftools简介 SAMtools是一个用于操作sam和bam文件的工具合集，包含有许多命令。. 98% : N/A) # 总体上reads的匹配率 20000 + 0 paired in sequencing # 有多少reads是属于paired reads 10000 + 0 read1 Although I know how to get total number of mapped read using samtools flagstat (samtools flagstat file_sorted. The default output for FASTA and FASTQ formats include one base per non-gap consensus. fasta samtools faidx ref. paired - number of paired reads, mapped or unmapped, that are neither secondary nor supplementary (flag 0x1 is set and flags 0x100 (256) and 0x800 (2048) are not set). Sep 15, 2020 · 比对结束后，需要了解比对结果的情况，可以采用samtools flagstat进行统计. - sam与bam互转:-S指定输入文件格式为sam，不加则为bam,-b指定输出格式 (默认输出sam) 过滤功能-F：后 May 21, 2013 · samtools flagstat yeast_chip. Description. mapped. 643253 X 2 = 1286506 # hisat2. counts the number of alignments for each FLAG type. 运用相应的命令并正确解读输出信息，可以在测序分析过程中发挥重要作用。. 16 * 4), which is binary for 1000000, corresponding to the read in the first read pair. 69% : N/A) #比对到参考基因组上的reads数量 186737458 + 0 paired in samtools-flagstat - Man Page. samtools flagstat는 SAM/BAM 파일에 수록된 read alignment 정보를 출력해 주는 매우 유용한 프로그램이다. 0x400 bit set. Here's how to run samtools flagstat and both see the output in the terminal and save it in a file – the samtools flagstat standard output is piped to tee, which both writes it to the specified file and sends it to its standard output: samtools flagstat - counts the number of alignments for each FLAG type. 14608455 + 0 in total (QC-passed reads + QC-failed reads) ## reads总数 37967 + 0 secondary ##出现比对到参考基因组多个位置的reads数 0 + 0 supplementary samtools-flagstat - counts the number of alignments for each FLAG type. macs2 callpeak -t PrimaryKeratinocytes. Generate consensus from a SAM, BAM or CRAM file based on the contents of the alignment records. 9033398 # excluding supplementary and secondary reads filtered sequences: 0 sequences: 9033398 is sorted: 1 1st fragments: {"payload":{"allShortcutsEnabled":false,"fileTree":{"multiqc/modules/samtools":{"items":[{"name":"__init__. sam out. sam是最常见的存放mapping数据的格式，各种组学分析只要存在mapping Nov 19, 2017 · A BAM file is the binary version of a SAM file, a tab-delimited text file that contains sequence alignment data. bam) but I want to count total number of mapped read in a non-overlapping sliding window of fixed size (let's say 500 bp) only with respect to reference (hg19) (assigning each read only once by its start position). The first row of output gives the total number of reads that are QC pass and fail (according to flag bit 0x200). Save any singletons in a separate file. samtools flagstat repressed_C1_tagged_dedup2. 提取比对质量高的reads 目录. 0x400 bit set and neither 0x100 nor 0x800 bit set. bam files - they can be converted into a non-binary format ( SAM format specification here) and can also be ordered and sorted based on the quality of the alignment. Apr 15, 2024 · record data if it tried to remove an over-long tag. How to filter those reads? I usually use samtools to filter those reads. samtools 用了 secondary 的结果，所以比对率会稍微高一点. Sep 29, 2016 · If "samtools flagstat" doesn't output the number of secondary alignments (I don't recall the output off-hand), then the following will give you the number of primary alignments: Code: samtools view -cF 2308 alignments. bam secondary. bam 603909814 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 1723882 + 0 supplementary 7197218 + 0 duplicates 601809982 + 0 mapped (99. counted. 15% : N/A) 193108 Dec 21, 2018 · Usage: samtools flagstat [options] <in. EDIT: Yes, the results of sambamba flagstat you showed in your edited post indicate exactly what you said: 94. bam > test_first_pair. sam | head -5. py","path":"multiqc/modules/samtools/__init__. bam secondary：多重比对，有一个初始的比对，有0或多个secondary比对，在flag中表示为0x100 supplementary：不具有较大重叠的多个比对结果，一个拆成多个，其中一个作为代表性比对，其他作为supplementary，在flag中表示为0x800 samtools-flagstat - counts the number of alignments for each FLAG type. PS. highQual. samtools sort <bamfile> <prefix of sorted bamfile>. Samtools is designed to work on a stream. Output: samtools-flagstat - counts the number of alignments for each FLAG type. samtools view -q 255 | samtools flagstat -. samtools view aligned_reads. 👍 1. It seems sambamba doesn't account for secondary alignments, and then overestimates 'paired in sequencing' by May 22, 2014 · Sorted BAM files are indexed ( samtools index) Each step above can be done with commands below. Jul 21, 2019 · You could also turn them into fprintf so you can have a -o or --output option to set the output file name. After having completed this chapter you will be able to: Use samtools flagstat to get general statistics on the flags stored in a sam/bam file; Use samtools view to: compress a sam file into a bam file; filter on sam flags; count alignments; filter out a region; Use samtools sort to sort an alignment file based on samtools flagstat output/NA12878. 09% unmapped: The SN section contains a series of counts, percentages, and averages, in a similar style to samtools flagstat, but more comprehensive. Using -O tsv selects a tab-separated values format that can easily be imported into spreadsheet software. 0x100 bit set supplementary. SamTools: Flagstat¶ SamToolsFlagstat · 1 contributor · 2 versions. After having completed this chapter you will be able to: Use samtools flagstat to get general statistics on the flags stored in a sam/bam file; Use samtools view to: compress a sam file into a bam file; filter on sam flags; count alignments; filter out a region; Use samtools sort to sort an alignment file based on Mar 25, 2024 · samtools flagstat maps/bt2. bam> $ samtools flagstat a. DESCRIPTION Does a full pass through the input file to calculate and print statistics to stdout. Nov 20, 2023 · Introduction to Samtools: Samtools is a versatile suite of tools widely used in bioinformatics for manipulating and analyzing SAM/BAM files containing aligned sequencing reads. $ samtools view -q <int> -O bam -o sample1. As we have seen, the SAMTools suite allows you to manipulate the SAM/BAM files produced by most aligners. 3 Aug 11, 2023 · The flags that make a read not primary are (from the documentation) either 0x100 (SECONDARY) or 0x800 (SUPPLEMENTARY). primary duplicates. 0x2. May 18, 2014 · samtools flagstat <bamfile> prints out a host of useful stats about your mapping results, such as reads mapped. both 0x1 and 0x2 bits set and 0x4 bit not set with itself and mate mapped. bam > output. bam generated. Each FLAGS argument may be either an integer (in decimal, hexadecimal, or octal) representing a combination of the listed numeric flag values, or a comma-separated string NAME, ,NAME representing a combination of the flag names listed below. samtools index <sorted bamfile>. CRAM comparisons between version 2. bam. ) Notice: Samtools mpileup VCF and BCF output (deprecated in release 1. both 0x1 and 0x40 bits set. paired-end (or multiple-segment) sequencing technology. samtools view -h -f 0x0040 test. cd samtools_exercise. 0 and BAM formats. Output paired reads in a single file, discarding supplementary and secondary reads. 0x800 bit set. Each category in the output is broken down into QC pass and QC fail. Both of these tools can be installed using conda if needed. bam 20000 + 0 in total (QC-passed reads + QC-failed reads) # 总共的reads数 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 18995 + 0 mapped (94. 62. i have ~26 million high quality paired end reads each pair 1 and pair2. $ samtools flagstat ENCFF653FOQ. 40% : 52. samtoolsflagstat是SAMtools软件包中非常常用的工具之一，能够方便地统计不同标志位的测序数据，获取测序数据的重要质量指标，并进行数据分析和后续的数据处理。. bz2 . view view命令的主要功能是查看bam和sam文件的内容。. Only one of the multiple read alignments is considered primary, and this decision may be arbitrary. sorted. The commands below are equivalent to the two above. see also: → Calling SNPs/INDELs with SAMtools/BCFtools. samtools flagstat로 출력되는 숫자를 재현하기. then i cheked it with samtools and took stats with samtools flagstat it show the result mentioned below: Aug 15, 2009 · SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. g. 对sam文件的操作是基于对sam文件格式的理解：. samtools flagstat in. Mar 26, 2020 · According to samtools documentation for flagstat: Provides counts for each of 13 categories based primarily on bit flags in the FLAG field. duplicates. Note: SAMtools mpileup counts only primary aligned reads. It can print a lot more information like % properly paired, # of duplicates, but it's simply relying on the information encoded in the second field of the SAM file - the bitwise flag field. i mapped it on reference genome with tophat and output file accepted_hits. 65% : N/A) 194412 + 0 paired in sequencing 97206 + 0 read1 97206 + 0 read2 190812 + 0 properly paired (98. bam | in. 92%) 3288632 + 14722 paired in sequencing 3288632 + 14722 read1 0 + 0 read2 3269034 + 7714 properly paired (99. 64000; now it is set to over 2 billion, so effectively all bases will be. 0x400 bit set and neither 0x100 nor 0x800 bit set mapped. So, in the above example, the total number of reads (R1+R2) should be 192013910 - 38830640. The encoded properties will be listed under Summary samtools flagstat - counts the number of alignments for each FLAG type. read1. 33% : N/A) 25619466 + 0 paired in sequencing 12809733 + 0 read1 12809733 + 0 read2 24045298 + 0 properly paired (93. (The "Source code" downloads are generated by GitHub and are incomplete as they don't bundle HTSlib and are missing some generated files. Simple stats using SAMTools flagstat. For example: 122 + 28 in total (QC-passed reads + QC-failed reads) Which would indicate that there are a total of 150 Jan 26, 2021 · Use samtools flagstat with option -O tsv:. Nov 4, 2020 · The samtools flagstat only check the FLAG, not the read ID. cram. This tutorial will guide you through essential commands and best practices for efficient data handling. bam -g hs -n test --nomodel --shift -51 --extsize 102 --q 0. 00% : N/A) 2030516 + 0 paired in sequencing 1015258 + 0 read1 1015258 We would like to show you a description here but the site won’t allow us. 25730940 + 0 in total (QC-passed reads + QC-failed reads) 111474 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 25300974 + 0 mapped (98. Application error: a client-side exception has occurred (see the browser console for more information). All the other alignments have the secondary alignment flag set in the SAM records that represent them. 这样两个结果就可以联系起来了. Previously this limit was set at. (PR #1758, fixes samtools/samtools#2002) limit used when calculating the pileup. The consensus is written either as FASTA, FASTQ, or a pileup oriented format. bam文件是sam文件的二进制 Feb 19, 2022 · 双端测序数据用samtools rmdup效果很差，很多人建议用picard工具的MarkDuplicates功能。. global. There are many sub-commands in this suite, but the most common and useful are: Convert text-format SAM files into binary BAM files ( samtools view) and vice versa. 0x1 bit set read1. View 作用：bam与sam互转，查看bam文件，对bam进行排序和提取的操作。. primary mapped. this should agree with the number of unique reads. namesorted. samtools view -bS <samfile> > <bamfile>. Note that "-F 2304" and "-F 0x2304" are very different things. Viewing and Filtering BAM Files: View a BAM file: bashCopy code samtools view file. Mar 24, 2022 · I wonder if someone please explain what secondary, supplementary, duplicates and paired in sequencing mean in samtools flagstat. 00% : 77. DESCRIPTION ¶. SYNOPSIS secondary 0x100 bit set supplementary 0x800 bit set duplicates The Samtools module parses results generated by Samtools, a suite of programs for interacting with high-throughput sequencing data. tar. bam] -q 设置 MAPQ (比对质量) 的阈值，只保留高于阈值的高质量 The output file is suitable for use with bwa mem -p which understands interleaved files containing a mixture of paired and singleton reads. e. 특히 하나의 read가 We will use samtools to view the sam/bam files. 2 - February 2015 ). or primary only alignments. 0x1 bit set and neither 0x4 nor 0x8 bits set Samtools Learning outcomes. samtools的rmdup是直接将这些重复序列从比对BAM文件中删除掉，而Picard的MarkDuplicates默认情况则只是在BAM的FLAG信息中标记出来，而不是删除，因此这些重复序列依然会被留在文件中 Oct 28, 2019 · Example: samtools depth aln. SYNOPSIS¶ samtools flagstat in. Jul 25, 2023 · secondary. read2. bam samtools idxstats aln. fasta samtools fasta input. 05 --keep-dup 'all' macs2 Jun 7, 2023 · Overview. bam; idxstats-- BAM index stats 统计bam索引文件里的比对信息 $ samtools idxstats Usage: samtools idxstats <in. SYNOPSIS ¶. 0x4 bit not set paired in sequencing. 00% : N/A) 2030511 + 0 primary mapped (100. bam > all_reads. fastq samtools head in. 0x400 bit set primary duplicates. 一、原理解析 . Sep 27, 2020 · Try samtools flagstat or sambamba flagstat to get similar type of information about your bam file. A joint publication of SAMtools and BCFtools improvements over the last 12 years was published in 2021. 0x1 bit set. Jan 4, 2022 · There is no flag for directly selecting the primary alignments - instead you need to remove the SECONDARY, the SUPPLEMENTARY, and the UNMAPPED alignments to be left with the so-called primary alignments. Copy an exercise SAM file to your SCRATCH directory. Retrieve and print stats in the index file corresponding to the input file. samtools-flagstat - counts the number of alignments for each FLAG type. bam> 命令：samtools flagstat mybam 结果解析： 3826122 + 0 in total (QC-passed reads + QC-failed reads) #总共的reads数 0 + 0 secondary 1658 + 0 supplementary 343028 + 0 duplicates 3824649 + 0 mapped (99. fq. bam 187018343 + 0 in total (QC-passed reads + QC-failed reads) #总reads数 280885 + 0 secondary #不知道 0 + 0 supplementary #不知道 0 + 0 duplicates #重复reads的数量 186439767 + 0 mapped (99. 65% : N/A) 602185932 + 0 paired in sequencing 301084296 + 0 read1 301101636 + 0 read2 590992186 + 0 properly paired (98. 28. 9) has been removed. DESCRIPTION¶ Does a full pass through the input file to calculate and print statistics to stdout. Jan 7, 2014 · I have used BWA 0. sam为bam文件的十进制文件；bam为二进制文件。. 以下内容整理自【直播我的基因组】系列文章. mkdir samtools_exercise. 91% = 5. Which in turn, cannot can not read the header of the input file "20201032. It's also indicated by "paired in sequencing", 153183270 (R1 +R2). I have run this pipeline many times before with no problems, but this one genome had presented something I haven't seen before. sam|sample1. samtools fastq -0 /dev/null in_name. bam samtools fqidx ref. After having completed this chapter you will be able to: Use samtools flagstat to get general statistics on the flags stored in a sam/bam file; Use samtools view to: compress a sam file into a bam file; filter on sam flags; count alignments; filter out a region; Use samtools sort to sort an alignment file based on Samtools is a set of utilities that manipulate alignments in the SAM (Sequence Alignment/Map), BAM, and CRAM formats. 3318824 + 14722 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 3318824 + 11471 mapped (100. 5. PCR duplication = 4th row / 1st row = 71809672 / 253552402 = 0. 0x0040 is hexadecimal for 64 (i. 75a to map PE data of WGS to reference, and samtools flagstat to check the resulting BAM file. 0x400 bit set mapped. Publications Software Packages. 2nd calculation produces the duplication rate very close to what is reported in Zlib implementations comparing samtools read and write speeds. Samtools Learning outcomes. 86% : N/A) DESCRIPTION. Below is the command am using and the outputs for macs and samtools flagstat. new. All the SAM records have the same QNAME and the same values for 0x40 and 0x80 flags. (PR #1970, fixes #1950. 1 manual page. @sq SN:2 LN:250202058. To decode a given SAM flag value, just enter the number in the field below. bam; flagstat-- simple stats 统计bam文件中read的比对情况 $ samtools flagstat Usage: samtools flagstat [--input-fmt-option OPT=VAL] <in. This is selected using the -f FORMAT option. both 0x1 and 0x80 bits set. reads duplicated - number of duplicate reads (flag 0x400 (1024) is set). The flagstat command provides simple statistics on a BAM file. Jul 15, 2018 · Charpter_14 Sequence Alignment Maps (SAM) SAM文件是由Tab (\t)分割，面向‘行’的文本格式文件，主要包括两部分：. Download the source code here: samtools-1. The header of the sam file looks as follows: @sq SN:1 LN:278617202. Example: 194492 + 0 in total (QC-passed reads + QC-failed reads) 80 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 193804 + 0 mapped (99. For more details on each argument, see the list further down below the table or click on an argument name to jump directly to that entry in the list. both 0x1 and 0x40 bits set read2. cds. bam> Example: samtools flagstat aln. We’ll use the samtools view command to view the sam file, and pipe the output to head -5 to show us only the ‘head’ of the file (in this case, the first 5 lines). secondary. raw total sequences - total number of reads in a file, excluding supplementary and secondary reads. Hi, thanks for putting together an excellent toolkit, the multithreading is a real win! I ran samtools flagstat, and then sambamba flagstat, and noticed that they handle secondary alignments differently. Tabs and trailing spaces are not allowed. samtools flagstat统计bam文件比对后每一个参数的解释如下：. 1. dup. bam aln. 15. Please use bcftools mpileup instead. In the default output format, these are presented as "#PASS + #FAIL" followed by a description of the category. 0x800 bit set duplicates. both 0x1 and 0x80 bits set properly paired. Mapping tools, such as Bowtie 2 and BWA, generate SAM files as output when aligning sequence reads to large reference sequences. 1, version 3. (PR #1752, PR #1761) CRAM files. 그러나 출력되는 정보는 기본적으로 read가 아닌 'alignment'라서 직관적으로 이해하기 어려울 때가 많다. You can count unique only alignments with. py samtools是一个用于操作sam和bam文件的工具合集。. This utility makes it easy to identify what are the properties of a read based on its SAM flag value, or conversely, to find what the SAM Flag value would be for a given combination of properties. bam> samtools flagstat tmp. 5 SO:coordinate@SQ SN:ref LN:45r001 99 ref 7 30 8M2I4M1D3M = 37 39 TTAGATAAAGGATACTG *r002 0 ref The samtools flagstat tool provides a simple analysis of mapping rate based on the the SAM flag fields. Argument name (s) samtools flagstat Sample1-A_dup_merged. sam|in. Also, don't forget to add tests and update the samtools-flagstat. Feb 7, 2022 · FlagStat specific arguments. fastq samtools fixmate in. You can use samtools flags to tell you what a flag value means. Header部分，包括 @HD, @SQ, @PG 等开头的基本信息。. Typically the alignment designated primary is the best alignment, but the decision may be arbitrary. The encoded properties will be listed under Summary Aug 29, 2022 · step2 - sam2bam and flagstat in samtools to generate sorted, indexed bam and statistics: 0 0 secondary 0 0 supplementary 0 0 duplicates 0 0 primary duplicates We would like to show you a description here but the site won’t allow us. 0x100 bit set. bam 2032568 + 0 in total (QC-passed reads + QC-failed reads) 2030516 + 0 primary 2052 + 0 secondary 0 + 0 supplementary 7207 + 0 duplicates 7207 + 0 primary duplicates 2032563 + 0 mapped (100. Dec 16, 2022 · In this case samtools view and samtools index failed in open the file "20201032_sorted. PAIRED. Synopsis. bam [sample1. So samtools flags I have seen previous posts that mention using "--keep-dup 'all'" option but I have deduplicated my bam using picard before macs. DESCRIPTION. 14% : N/A) Dec 21, 2015 · Primary Alignment and Secondary Alignment: A read may map ambiguously to multiple locations, e. bam samtools flags PAIRED,UNMAP,MUNMAP samtools flagstat aln. Feb 3, 2021 · Here is my flagstat output after running bwa mem on my paired end data. sort. (If there is secondary or duplicate, it should also be excluded). samtools view --input-fmt cram,decode_md=0 -o aln. due to repeats. 00% : N/A) 0 + 0 paired in sequencing 0 + 0 read1 0 + 0 read2 0 + 0 properly paired (N/A : N/A) 0 + 0 with itself and mate mapped 0 + 0 singletons (N/A : N/A) 0 + 0 with mate Oct 19, 2017 · Usage: samtools flagstat <in. Argument name (s) Dec 3, 2018 · 1299533 - 13027 = 1286506 # samtools. 0x4, 0x100 and 0x800 bits not set. The head of a SAM file takes the following form:@HD VN:1. both 0x1 and 0x80 bits set properly paired Decoding SAM flags. properly paired Decoding SAM flags. Same number reported by samtools view -c -F 0x900. sam" . suf. The samtools idxstats prints its results to standard out (no consistent file name) and has no header lines (no way to recognise 1. bam Watch out for mapper-specific data Not all mappers actually write data to that bitfield, or may not populate a field if you don't instruct the mapper right; for example, if you have paired-end data but tell the mapper to map both reads as single-end reads, you won't get pairing stats. It may be easier to interpret. It is best written out explicitly like so: samtools flags -F 256 -F 2048 -F 4 Sep 16, 2022 · Hi, i don't undersand if my data are good and how interprete it. All other alignments have the secondary alignment flag. Does a full pass through the input file to calculate and print statistics to stdout. In this format the first column contains the values for QC-passed reads, the second column has the values for QC-failed reads and the third contains the category names. Note that samtools code uses four spaces for indentation. 0x4 bit not set primary mapped. We should make the documentation on flagstats clearer. bam 647063 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 647063 + 0 mapped (100. Also, all reads are presumed to be mapped unless they are marked with the UNMAP flag. 91% mapped and 100% - 94. both 0x1 and 0x80 bits set properly paired Nov 3, 2020 · samtools flagstat out. FLAGS: 0x1. My pipeline includes the usual sorting, fixing malformed bams, and marking duplicates. Sep 25, 2014 · the difference is due to multi-mappers, which samtools flagstat counts as separate "reads" (or rather alignments). Jul 25, 2023 · samtools dict-a GRCh38 -s "Homo sapiens" ref. sam. 96% : N/A) #总体reads的匹配率 3824464 + 0 paired in sequencing #总共的reads数 1912442 + 0 read1 #reads1中的reads数 1912022 May 17, 2017 · Samtools allows you to manipulate the . If run on a SAM or CRAM file or an unindexed BAM file, this command will still produce the same summary statistics, but does so by reading through the entire file. PCR duplication = 4th row / 9th row = 71809672 / 114967188 = 0. It converts between the formats, does sorting, merging and indexing, and can retrieve reads in any regions swiftly. Alignment部分，包括reads比对的所有信息。. We would like to show you a description here but the site won’t allow us. paired in sequencing. cram aln. 这些命令的使用方法如下： 1. samtools view -O cram,store_md=1,store_nm=1 -o aln. supplementary. Sep 17, 2021 · 0 + 0 with mate mapped to a different chr (mapQ>=5) To determine PCR duplication rate from the above values, I have two options. BCFtools主要是用来操作vcf和BCF文件的工具合集，包含有许多命令。. Before calling idxstats, the input BAM file should be indexed by samtools index. SAMtools discards unmapped reads, secondary alignments and duplicates. Provides counts for each of 13 categories based primarily on bit flags in the FLAG field. 两个结果都没有错，只是统计方法和策略不一样. Supported commands: stats; flagstats; idxstats; rmdup; coverage; markdup; idxstats. SYNOPSIS samtools flagstat in. bam View May 10, 2015 · drmjc commented on May 10, 2015. Sort BAM files by reference coordinates ( samtools sort) An alternative way of achieving the above is listing multiple options after the --output-fmt or -O option. samtools 操作指南. bam|in. sam" , because this file should be the output of samtools sort. kk wc me jp bm cs ro wb ss ls