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Samtools flagstat mapped reads

Samtools flagstat mapped reads. → How to count the number of mapped reads in a BAM or SAM file (SAM bitcode fields) more statistics about alignments. For example: 32 + 0 mapped (94. 5512 ( 1. 281. 708109 + 0 mapped (0. sam > output. 76%) 14745930 with mate mapped to a You signed in with another tab or window. Aug 18, 2011 · If that bit flag is not set then the read is mapped. reads QC failed - number of reads that failed Count the number of reads mapped on chromosomeIII samtools view -F 0X04 <bamfile> chrIII | wc -l Extract/print reversely mapped sub alignments in BAM format If you have a strand-specific RNA-seq data, a mapping direction of a read is critical. 7% overall read mapping rate. flagstat Since the BAM file contains all the information from the original SAM file, remember to remove the SAM file once you are finished, in order to free up disk space. Mar 16, 2020 · When I used samtools flagstat to check these . fai； samtools tview：将比对结果reads与基因组显示在同一张图中，类似于基因组浏览器； samtools flagstat：给出bam的比对结果； [Samtools-help] How samtools flagstat report a read mapped or not? Brought to you by: awhitwham , bhandsaker , daviesrob , jenniferliddle , and 5 others Summary SAMTOOLS FLAGSTAT. e. reads QC failed - number of reads that failed The top-level object contains two named objects QC-passed reads and QC-failed reads. You can count unique only alignments with. 69% : N/A) 1286506 + 0 paired in sequencing 643253 + 0 read1 643253 + 0 read2 805670 + 0 properly paired (62. These are values for trimmed input. The reads map to multiple places on the genome, and we can't be sure of where the reads originated. After trimming and filtering my reads I am left with The output is TAB-delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads. 3. How to count the number of mapped reads in a BAM or SAM file? # get the total number of reads of a BAM file (may include unmapped and duplicated multi-aligned reads) samtools view -c SAMPLE. 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 Jun 19, 2017 · samtools flagstat -@ 4 sample1. 그러나 출력되는 정보는 기본적으로 read가 아닌 'alignment'라서 직관적으로 이해하기 어려울 때가 많다. samtools flagstat统计bam文件比对后每一个参数的解释如下：. 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). SamToolsFlagstat · 1 contributor · 2 versions. Use samtools to create a flagstat file from a bam or sam file. samtools flagstat로 출력되는 숫자를 재현하기. youtube. flagstat. samtools flagstat는 SAM/BAM 파일에 수록된 read alignment 정보를 출력해 주는 매우 유용한 프로그램이다. bam> Example: samtools flagstat aln. 91. 3%) are discordant alignments. samtools view -b -F 4 file. 结果解释. bam 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. In order to extract mapped reads from a bam file, use -F option in samtools view command. Mapping qualities are a measure of how likely a given sequence alignment to a location is correct. samtools view -c . Having ref aligned data and previously removing reads that don't map I now want to produce a single file with the remaining number of mapped read outputs into a single file. 由约翰霍普金斯大学计算生物学中心（CCB at JHU）开发，是TopHat的升级版本，速度提高了50倍。. 42% : N/A) 21422330 + 0 with itself and mate mapped 0 + 0 singletons (0. For example: You can also get a report on your mapped reads using the samtools command flagstat: samtools flagstat properName. The samtools flagstat tool provides a simple analysis of mapping rate based on the the SAM flag fields. It is flexible in style, compact in size, efficient in random access and is the format in which alignments When I use samtools flagstat to see the no. 00% : N/A) 1306000 + 0 Jan 29, 2010 · samtools flagstat mismatch in stats. sam" samtools sort: failed to read header from "20201032. In this section we will use our skill on the command-line interface to map our reads from the evolved line to our ancestral reference genome. Save any singletons in a separate file. 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. Note that if you map with BBMap (which also adds XT:A:U and XT:A:R tags), you can use the flag "ambig=toss Jun 4, 2012 · 1. sam | head -5. Note. Dhahbi, PhD Dec 28, 2011 · 72008 + 0 with mate mapped to a different chr (mapQ>=5) I don't understand the detailed explanation of samtools flagstat output. Aug 9, 2011 · I got the following info after running a samtools flagstat on a Novoalign bam file: 126597089 in total 0 QC failure 0 duplicates 122446987 mapped (96. Sep 17, 2022 · Full tutorial with explanation: https://www. Since the duplication marking does not look at the unmatched reads making a metric based on this probably won't give you useful information. Before calling idxstats, the input BAM file should be indexed by samtools index. Exercise 3 Count the number of reversely-mapped reads overlapping between chromosome III: 123456 and chromosome III: 124456 Hint: flag 0x10 = SEQ being reverse complemented May 17, 2017 · Filtering bam files based on mapped status and mapping quality using samtools view. samtools flagstat in. 19%) 118953502 with itself and mate mapped 3493485 singletons (2. 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 Jul 8, 2020 · 所以进一步查了一下为什么不是proper pair。. 0x0040 is hexadecimal for 64 (i. Mar 4, 2010 · The author (s) (Heng) specifically recommends moving away from samtools rmdup and use picard. 0 + 0 duplicates. 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。. DESCRIPTION ¶. get number of reads. bam files, I notice that most reads were unmapped. comprehensive statistics. of these: 211726 (48. It's already relatxed - that's part of the problem. 74%) can you please explain the meaning of '13950954 in total'? Regards, Joseph. samtools flagstat : Simple stats about how many reads mapped to the reference, how many reads were paired properly etc. Running samstat on BAM files. samtools flagstat SAMPLE. The original fastq files with the reads had a size of ~1. From the manual; there are different int codes you can use with the parameter f, based on what you For example, you may want to count only reversely-mapped reads on a (-)-direction gene. 34%). The lowest score is a mapping quality of zero, or mq0 for short. We can perform a basic assessment of the read alignment for all our samples with using the samtools flagstat command: Samtools Learning outcomes. 16 * 4), which is binary for 1000000, corresponding to the read in the first read pair. Sep 7, 2010 · When I used samtools to look at the bam file, it gave me 13950954 in total; but I don't see this number in the above bowtie output: samtools flagstat myFile. It is a contig of only 1 read. samtools flagstat . This value is already parsed by MultiQC and can be We would like to show you a description here but the site won’t allow us. . inputs. May 21, 2013 · If you use bwa with default options, the output bam includes unmapped reads. In samtools, a singleton refers to a read that mapped but the mate didn't. Mapping stats with SAMtools. bam文件是sam文件的二进制 Jul 24, 2014 · Now let's get started! For the first test, I took some sequence from the human genome (hg19) and created two 100 bp reads from this region. 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). These contain the various categories listed above as names and the corresponding count as value. 3153604 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 18472 + 0 mapped (0. Oct 22, 2014 · There's two interpretations: Model A: primary and secondary alignments are independent sets of alignments. Ensure all reference files are available: Note. samtools stats SAMPLE. bam [bam_header_read] EOF marker is absent. 从第一行至第十一行分别表示：. info. bam > mapped. bam > properName. For example: 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. 01-29-2010, 10:54 AM. For example, you may want to count only reversely-mapped reads on a (-)-direction gene. # counting only mapped (primary aligned) reads. The out. This directly contradicts what you posted in the prep_reads. 41721001 mapped (93. Generate user input files for SamToolsFlagstat: # user inputs. fq. 21611397 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 21611397 + 0 mapped (100. 66% : N/A) 9860 + 0 paired in sequencing 4950 + 0 read1 4910 + 0 read2 9611 + 0 properly paired (97. 如图所示，这这两种方式的时候，reads是proper paired的，即 forward + read1和reverse + read2； 或者 forward + read2和reverse + read1。. SEE ALSO: Aug 15, 2009 · Abstract. samtools view -c -F 260 SAMPLE. bai； samtools faidx：为FASTA文件建立索引，索引文件的后缀为. -F INT means "Skip alignments with bits present in INT". Exercise: Check out the flag that you would need to filter for mapped reads. To assess the total % of mitochondrial reads, samtools idxstats can be run to report the total number of reads mapped to each chromosome. Flagstat says about 40% of my reads are singletons, but I feel like based on the 'view' command I used, they should ALL be singletons. 12% : N/A) The properly paired and singletons counts work in a similar way but the percentage is against the total number of QC-passed and QC-failed pairs. Jun 9, 2023 · Since the BAM file contains records for both mapped and unmapped reads, just counting records doesn't provide information about the mapping rate of our alignment. or primary only alignments. 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 The SN section contains a series of counts, percentages, and averages, in a similar style. You switched accounts on another tab or window. samtools fastq -0 /dev/null in_name. bam > eg/first. bam > sample1_flagstats samtools flagstat -@ 4 sample2. bam|in. Once again, you can use flags to verify this (it also accepts hexadecimal input). Same number reported by. Write the solutions and answers into a text-file. 66% : N/A) 9826 + 0 primary mapped (99. Hi, I'm working with RNA-Seq and using bowtie and tophat to align 65bp PE reads to a reference genome. In other words, -F INT filters reads that have the INT in their flag. bam > unmapped. sam SamTools: Flagstat¶. to samtools flagstat, but more comprehensive. bam 1299533 + 0 in total (QC-passed reads + QC-failed reads) 13027 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 866610 + 0 mapped (66. 1. 72%) 126597089 paired in sequencing 63372478 read1 63224611 read2 104053862 properly paired (82. Feb 22, 2017 · 94. 93% : N/A) 76124384 + 0 paired in sequencing. both_mates_unmapped. samtools view aligned_reads. The input bam file must be sorted and indexed. Enhanced Data Retrieval with Samtools Index: Quick Access to Genomic These should be removed. Mean Read Depth Ensure Janis is configured to work with Docker or Singularity. Table of Contents. sam output from bwa mem was converted to bam, sorted and indexed; the duplicates were marked with the command MarkDuplicates from picard; Then if I call samtools flagstat on the sorted bam file which had the duplicates marked with picard I get: We will use samtools to view the sam/bam files. 12% : N/A) In the tsv and json formats, these percentages are listed in separate categories mapped %, properly paired %, and singletons %. Extract/print sub alignments in BAM format. Answer. sam" : No such file or directory samtools view: failed to The top-level object contains two named objects QC-passed reads and QC-failed reads. bam; idxstats-- BAM index stats 统计bam索引文件里的比对信息 $ samtools idxstats Usage: samtools idxstats <in. this should agree with the number of unique reads. 38062192 + 0 read2. Code: # samtools view -F 4 -b -o <out. Mapping stats with samstat. bam. 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. Mar 26, 2020 · aligned the fastq reads against the reference genome with bwa mem; the . Aligned pairs: 433356. 98% : N/A) 213393 + 0 with mate mapped to a different chr. However, its 0 (zeros) for all. 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 + 0 Jun 12, 2015 · In general genomic assembly, a singlton is a read that did not assemble into a contig or map to a reference. Mar 29, 2023 · $ samtools flagstat _. 1 utilities depth and flagstat. 出力結果の例. Although I definitely have 12 million aligned reads according to bowtie2 and samtools flagstat. com/playlist?list=PLe1-kjuYBZ06jn12R4hiid7QGLqXWSJc4samtools flagstatSupport my work https://www. sam 9908 + 0 in total (QC-passed reads + QC-failed reads) 9860 + 0 primary 0 + 0 secondary 48 + 0 supplementary 0 + 0 duplicates 0 + 0 primary duplicates 9874 + 0 mapped (99. 82%) Jul 27, 2022 · mapping法评估基因组组装质量。mapping法是指把测序的reads（包括Pacbio，Illumina，RNA-seq 等reads）映射回组装好的基因组，评估mapping rate，genome coverage，depth分布等指标，用这些指标评估基因组组装质量。这篇文章简单介绍了mapping法的其中一个评估指标：mapping rate。通过samtools计算mapping r Dec 2, 2019 · To give an idea of the extent of the problem. Oct 28, 2019 · Example: samtools depth aln. Here, the options are: -b - output BAM, -f12 - filter only reads with flag: 4 (read unmapped) + 8 (mate unmapped). When fastq files are generated with the command above, these files have a size in the order of 70MB. Instead of using the '-f' required flag parameter with samtools view use the '-F' filter flag parameter to remove unmapped reads. 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. Model B: primary and secondary alignments jointly form a graph of alignments. 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 Sep 17, 2021 · Both Picard and samtools markdup both work in a similar way by comparing mapped position and orientation of reads and seeing if they match. 568345. 利用 HISAT2 + StringTie 流程，可以快速地分析转录组测序数据，获得每个基因和转录本的表达量。. Jul 29, 2020 · To extract only the reads where read 1 is unmapped AND read 2 is unmapped (= both mates are unmapped): samtools view -b -f12 input. Output: Samtools flagstat - low % reads mapping. bam > sample3_flagstats multiqc . Preface ¶. 11-01-2012, 06:35 AM. Mar 26, 2019 · An alignment of RNA-seq reads is usually considered to have succeeded if the mapping rate is >70%. samtools sort: failed to read header from "20201032. laevis and I'm attempting to first map to X. The reads were then mapped back to the reference using BWA aln and sampe. Converting SAM files to BAM files. The 256 bit (0x100) indicates that an alignment is secondary, which is relevant, but won't help you in this case. 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。. Additional SAMtools tricks. Does a full pass through the input file to calculate and print statistics to stdout. Directionality of mapped read is also recorded on flag. * What does "singleton" mean ? This means that one reads of the paired end reads is mapped to the genome * What "with itself and mate mapped" mean? It means that both reads mapped to genome Apr 4, 2016 · 2871947 + 0 with itself and mate mapped. Decoding SAM flags. sorted. Let’s take a look at the first few lines of the original file. 3GB. For example: DESCRIPTION. 5. My reads were sequenced from X. Provides counts for each of 13 categories based primarily on bit flags in the FLAG field. 9%) have multiple alignments. laevis genome is still draft version). Apr 29, 2021 · 准备序列比对后生成的 bam 文件或者 . For the default format, mapped shows the count as a percentage of the total number of QC- passed or QC-failed reads after the category name. The top-level object contains two named objects QC-passed reads and QC-failed reads. 1. 00% : N/A) 21422330 + 0 paired in sequencing 10711165 + 0 read1 10711165 + 0 read2 19797684 + 0 properly paired (92. tax. Read mapping ¶. 3% concordant pair alignment rate. samtools view -b -f 0x0040 eg/ERR188273_chrX. 3. yaml. May 18, 2014 · Theory. Reload to refresh your session. of read mapped to the genome I get a lot of warnings like: [W::sam_parse1] unrecognized reference name; treated as unmapped as explained in another Biostar question, this happens whenever you have a read mapping to a chromosome/contig that's not in the header but in my case as can be seen in output. bam > sample2_flagstats samtools flagstat -@ 4 sample3. 213393 + 0 with mate mapped to a different chr (mapQ>=5) Two things The top-level object contains two named objects QC-passed reads and QC-failed reads. buyme 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。. When I try to get the number of aligned reads 2 different ways I get 2 different numbers When I run flagstat: samtools flagstat myBam. 1st row includes all reads, matched and unmatched. bam> <in. Apr 26, 2018 · #命令解析 $ samtools flagstat sample1. bam Mar 19, 2018 · However you can show how many non-zero entries there are easily enough: I need a read count per chromosome and I'm using samtools idxstats. 19% vs 99. raw total sequences - total number of reads in a file. The encoded properties will be listed under Summary Description of feature Hello, I would like to display the percentage of mapped reads given by samtools flagstat results in the general stat table. 4GB in size each, after alignment (bwa mem + samtools sort) the resultant BAM file had a size of ~2. samtools idxstats : This tool will provide statistics about how many reads have aligned to each sequence/chromosome in the reference genome. For example: to get the output in bam, use: samtools view -b -f 4 file. I get this many mapped reads. 62% : N/A) 812960 + 0 with itself and mate Although I know how to get total number of mapped read using samtools flagstat (samtools flagstat file_sorted. This wrapper can be used in the following way: The output is TAB-delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads. sam" srun: error: node2-069: tasks 0-3: Exited with exit code 1 [E::hts_open_format] Failed to open file "20201032_sorted. janis inputs SamToolsFlagstat > inputs. Joseph M. I get zero aligned and zero unaligned reads as output. For example: 122 + 28 in total (QC-passed reads + QC-failed reads) Which would indicate that there are a total of 150 For the default format, the mapped, properly paired, and singletons show the count as a percentage of the total number of QC-passed or QC-failed reads after the category name. 특히 하나의 read가 Jan 4, 2012 · The total reads and mapped reads look good. bam: bam. cram. You will need the 0x4 flag. It must has positive numbers. Without trimming, I get 0% properly paired reads in samtools flagstat after snap paired mappi 5. 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. Hi all, I have a bam file created from illumina alignments with bwa. You will encounter some To-do sections at times. tropicalis (X. sam|in. filtered sequences - number of discarded reads when using -f or -F option. 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). Output files of mapping. Filtering against unmapped reads (leaving only mapped reads) with samtools view would look like this: May 17, 2017 · Filtering bam files based on mapped status and mapping quality using samtools view. More information about these inputs are available below. 重复reads的数量，QC pass和failed. Watch out for mapper-specific data. You signed out in another tab or window. I marked the duplicates by picard, samtools flagstat can detect the number of duplicates, but still cannot find the number of QC failure. 13950954 in total 12240725 mapped (87. 0 + 0 supplementary. TopHat采用外显子 Sometimes you only want the first pair of a mate. The first row of output gives the total number of reads that are QC pass and fail (according to flag bit 0x200). The inner mate distance between the two reads is 200 bp, creating an insert size of 400 bp. Samtools pileup with -m [1796] is said to have the ability to filter out QC failure and duplicates. bam file will contain only reads which mapped, regardless of whether their mates mapped or not. For example: Nov 19, 2017 · The mean read depth, the breadth of coverage of the reference genome, and the proportion of the reads that mapped to the reference genome can be obtained from a BAM file using the combination of awk, and the SAMtools 1. It is written to stdout. Extract/print mapped sub alignments in BAM format. bam>. Samtools是一款操作序列比对的实用软件，本文介绍了它的基本功能和使用方法，适合生物信息学的初学者和研究者。 samtools index：为排序后的bam文件建立索引，索引文件的后缀为. bam > all_reads. Jun 16, 2023 · HISAT2 是一款利用改进的BWT算法进行序列比对的软件。. 59% : N/A) Hi all, I have a series of SAM files, which I know that I have to convert to BAM and sort for using FLAGSTAT and VIEW in samtools. sam 文件. get number of individual reads, paired reads that mapped both count double R1+R2. 2. 2. samtools view -c SAMPLE. Strategically Using Samtools Flagstat: Comprehensive Alignment Metrics: Obtain comprehensive alignment metrics with samtools flagstat. Retrieve and print stats in the index file corresponding to the input file. reads MQ0 - number of mapped reads with mapping quality 0. Jul 25, 2023 · The output is TAB-delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads. bam文件 > flagstat. 76124692 + 0 in total (QC-passed reads + QC-failed reads) 308 + 0 secondary. 38062192 + 0 read1. The reason I think we should relax it and make this the aligner's choice. 我在构建数据的时候，误把reverse的read放到了forward的read的左边了，直接bwa aln的时候就会出错 Nov 20, 2023 · 280. Hi, I am getting low % of properly paired reads in snap paired comparatively to bwa mem (24. 47% : N/A May 22, 2014 · If you use bwa with default options, the output bam includes unmapped reads. To decode a given SAM flag value, just enter the number in the field below. Output paired reads in a single file, discarding supplementary and secondary reads. In the default output format, these are presented as "#PASS + #FAIL" followed by a description of the category. It’s at page 7 of the SAM documentation. 354837 + 0 singletons (10. reads duplicated - number of duplicate reads (flag 0x400 (1024) is set). For the default format, mapped shows the count as a percentage of the total number of QC-passed or QC-failed reads after the category name. Example. The output file is suitable for use with bwa mem -p which understands interleaved files containing a mixture of paired and singleton reads. 这些命令的使用方法如下： 1. samtools flagstat provides a short report including the total number of DNA reads as the first line (halve this number for the total number of fragments). One thing that might be sensible to do at some point is to filter out unmapped reads. Sep 25, 2014 · the difference is due to multi-mappers, which samtools flagstat counts as separate "reads" (or rather alignments). Apr 28, 2014 · bwa adds "XT:A:R" to to ambiguously-mapped reads and "XT:A:U" to uniquely-mapped reads, so filtering by that tag should work. samtools view -q 255 | samtools flagstat -. The very first QC of aligned reads should be to generally check the aligner’s output. Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. To get only the mapped reads use the parameter F, which works like -v of grep and skips the alignments for a specific flag. We would like to show you a description here but the site won’t allow us. view view命令的主要功能是查看bam和sam文件的内容。. This command provides valuable statistics, including the number of mapped reads, to assess the overall quality of the alignment. QC pass的reads的数量为2499971，未通过QC的reads数量为0，意味着一共有2499971条reads；. Here's how to run samtools flagstat and both see the output in the terminal and 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). This says your input to tophat is only ~460k read pairs. I think 'samtools flagstat' doesn't detect a 'flags' on a BAM file ? 0 paired in sequencing 0 read1 0 read2 0 properly paired (nan%) 0 with itself and mate mapped 0 singletons (nan%) 0 with mate mapped to a different chr Dec 3, 2018 · [will@200server Arb-hisat]$ samtools flagstat RNA. lz hl kb vt bt vd lg qh cl gn